U.S. patent application number 15/506643 was filed with the patent office on 2017-09-28 for trophoblast glycoprotein (5t4, tpbg) specific chimeric antigen receptors for cancer immunotherapy.
The applicant listed for this patent is CELLECTIS. Invention is credited to Cecile SCHIFFER-MANNIOUI.
Application Number | 20170275374 15/506643 |
Document ID | / |
Family ID | 51589045 |
Filed Date | 2017-09-28 |
United States Patent
Application |
20170275374 |
Kind Code |
A1 |
SCHIFFER-MANNIOUI; Cecile |
September 28, 2017 |
TROPHOBLAST GLYCOPROTEIN (5T4, TPBG) SPECIFIC CHIMERIC ANTIGEN
RECEPTORS FOR CANCER IMMUNOTHERAPY
Abstract
The present invention relates to Chimeric Antigen Receptors
(CAR) that are recombinant chimeric proteins able to redirect
immune cell specificity and reactivity toward selected membrane
antigens, and more particularly in which extracellular ligand
binding is a scFV derived from a 5T4 monoclonal antibody,
conferring specific immunity against 5T4 positive cells. The
engineered immune cells endowed with such CARs are particularly
suited for treating lymphomas and leukemia, and for solid tumors
such as colon, stomach, and ovarian tumors.
Inventors: |
SCHIFFER-MANNIOUI; Cecile;
(Villiers-sur-Marne, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CELLECTIS |
Paris |
|
FR |
|
|
Family ID: |
51589045 |
Appl. No.: |
15/506643 |
Filed: |
September 3, 2015 |
PCT Filed: |
September 3, 2015 |
PCT NO: |
PCT/EP2015/070128 |
371 Date: |
February 24, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/70517 20130101;
A61P 37/04 20180101; C07K 2319/02 20130101; C07K 14/7051 20130101;
C07K 2319/74 20130101; A61P 35/02 20180101; C07K 14/70535 20130101;
C07K 2317/622 20130101; C07K 2319/33 20130101; C07K 16/30 20130101;
C07K 19/00 20130101; C07K 2319/03 20130101; C12N 5/0634 20130101;
A61K 39/00118 20180801; A61P 35/00 20180101; C07K 14/70578
20130101; C12N 2510/00 20130101; C07K 2319/70 20130101; C07K 14/705
20130101; A61K 2039/5156 20130101; C12N 5/0636 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; C12N 5/0783 20060101 C12N005/0783; C07K 14/725 20060101
C07K014/725; C12N 5/078 20060101 C12N005/078; C07K 14/705 20060101
C07K014/705; C07K 19/00 20060101 C07K019/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 4, 2014 |
DK |
PA201470543 |
Claims
1-56. (canceled)
57. A 5T4 (NTRKR1) specific chimeric antigen receptor (CAR) having
a polypeptide structure V3, said structure comprising an extra
cellular ligand binding-domain comprising VH and VL chains from a
monoclonal anti-5T4 antibody, a CD8.alpha. hinge, a transmembrane
domain and a cytoplasmic domain including a CD3 zeta signaling
domain and a co-stimulatory domain from 4-1BB.
58. The 5T4 specific CAR according to claim 57, wherein said
structure V3 comprises a 4-1BB transmembrane domain or a CD8a
transmembrane domain.
59. The 5T4 specific CAR according to claim 57, wherein said
structure V3 comprises a CD8a transmembrane domain.
60. The 5T4 specific CAR according to claim 57, wherein said VH and
VL chains have at least 80% sequence identity with a polypeptide
sequence selected from SEQ ID NO. 11 to 17, or 18.
61. The 5T4 specific CAR according to claim 57, wherein said extra
cellular ligand binding-domain comprises a VH chain comprising the
CDRs from a mouse monoclonal antibody A1 of SEQ ID NO. 54 (CDR1),
SEQ ID NO.55 (CDR2) and SEQ ID NO.56 (CDR3), and a VL chain
comprising the CDRs from a mouse monoclonal antibody A1 of NO. 57
(CDR1), SEQ ID NO.58 (CDR2) and SEQ ID NO:59 (CDR3).
62. The 5T4 specific CAR according to claim 57, wherein said VH
chain has at least 80% sequence identity to SEQ ID NO:13 (A1-VH)
and said VL chain has at least 80% sequence identity to SEQ ID
NO:14 (A1-VL).
63. The 5T4 specific CAR according to claim 57, wherein said extra
cellular ligand binding-domain comprises a VH chain comprising the
CDRs from a mouse monoclonal antibody A2 of SEQ ID NO. 61 (CDR1),
SEQ ID NO.61 (CDR2) and SEQ ID NO.63 (CDR3), and a VL chain
comprising the CDRs from a mouse monoclonal antibody A2 of SEQ ID
NO. 64 (CD1), SEQ ID NO.65 (CD2) and SEQ ID NO:65 (CDR3).
64. The 5T4 specific CAR according to claim 57, wherein said VH
chain has at least 80% sequence identity to SEQ ID NO:15 (A2-VH)
and said VL chain has at least 80% sequence identity to SEQ ID
NO:16 (A2-VL).
65. The 5T4 specific CAR according to claim 57, wherein said extra
cellular ligand binding-domain comprises a VH chain comprising the
CDRs from a mouse monoclonal antibody A3 of SEQ ID NO. 66 (CDR1),
SEQ ID NO.67 (CDR2) and SEQ ID NO.68 (CDR3), and a VL chain
comprising the CDRs from a mouse monoclonal antibody A3 of SEQ ID
NO. 69 (CDR1), SEQ ID NO.70 (CDR2) and SEQ ID NO:71 (CDR3).
66. The 5T4 specific CAR according to claim 57, wherein said VH
chain has at least 80% sequence identity to SEQ ID NO:17 (A3-VH)
and said VL chain has at least 80% sequence identity to SEQ ID
NO:18 (A3-VL).
67. The 5T4 specific CAR according to claim 57, wherein said extra
cellular ligand binding-domain comprises a VH chain comprising the
CDRs from a mouse monoclonal antibody H8 of SEQ ID NO. 48 (CDR1),
SEQ ID NO.49 (CDR2) and SEQ ID NO.50 (CDR3), and a VL chain
comprising the CDRs from a mouse monoclonal antibody H18 of NO. 51
(CDR1), SEQ ID NO.52 (CDR2) and SEQ ID NO:53 (CDR3).
68. The 5T4 specific CAR according to claim 57, wherein said VH
chain has at least 80% sequence identity to SEQ ID NO:11 (H18-VH)
and said VL chain has at least 80% sequence identity to SEQ ID
NO:12 (H18-VL).
69. The 5T4 specific CAR according to claim 57, wherein
co-stimulatory domain from 4-1BB has at least 80 sequence identity
to SEQ ID NO.8.
70. The 5T4 specific CAR according to claim 57, wherein said CD3
zeta signaling domain has at least 80%, sequence identity with SEQ
ID NO. 9.
71. The 5T4 specific CAR according to claim 57, wherein said CD8a
hinge has at least 80% sequence identity with SEQ ID NO.4.
72. The 5T4 specific CAR according to claim 58, wherein said CD8a
transmembrane domain has at least 80% sequence identity with SEQ ID
NO.6.
73. The 5T4 specific CAR according to claim 58, wherein said 4-1BB
transmembrane domain has at least 80% sequence identity with SEQ ID
NO.7.
74. The 5T4 specific CAR according to claim 57, wherein the
polypeptide structure V3 comprises a polypeptide sequence having at
least 80% sequence identity to SEQ ID NO. 21, SEQ ID NO.27, SEQ ID
NO.33 or SEQ ID NO.39.
75. The 5T4 specific CAR according to claim 57, further comprising
a signal peptide.
76. A polynucleotide encoding a CAR according to claim 57.
77. An engineered immune cell expressing a 5T4 specific CAR
according to claim 57, wherein said 5T4 specific CAR is expressed
on the cell surface membrane.
78. An engineered immune cell according to claim 77, wherein said
cell is derived from inflammatory T-lymphocytes, cytotoxic
T-lymphocytes, regulatory T-lymphocytes or helper
T-lymphocytes.
79. A method of treating a disease or condition in a subject
comprising administering to the subject in need thereof a
therapeutic amount of the engineered cell according to claim
77.
80. The method according to claim 79, wherein the disease or
condition is a haematological cancer.
81. The method according to claim 79, wherein the disease or
condition is childhood pre-B acute lymphoblastic leukemia.
82. The engineered cell according to claim 77, wherein expression
of TCR is suppressed in said immune cell.
83. The engineered cell according to claim 77, wherein said cell is
mutated to confer resistance to at least one immune suppressive or
chemotherapy drug.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to Chimeric Antigen Receptors
(CAR) that are recombinant chimeric proteins able to redirect
immune cell specificity and reactivity toward 5T4, a cell surface
glycoprotein found on most myeloid cells and used to diagnose solid
tumors such as stomach, colon and ovarian tumors, and pre-B acute
lymphocytic leukemia (ALL) in patients. The CARs according to the
invention are particularly useful to treat malignant cells bearing
5T4 antigen, when expressed in T-cells or NK cells. The resulting
engineered immune cells display high level of specificity toward
malignant cells, conferring safety and efficiency for
immunotherapy.
BACKGROUND OF THE INVENTION
[0002] Adoptive immunotherapy, which involves the transfer of
autologous antigen-specific T cells generated ex vivo, is a
promising strategy to treat viral infections and cancer. The T
cells used for adoptive immunotherapy can be generated either by
expansion of antigen-specific T cells or redirection of T cells
through genetic engineering (Park, Rosenberg et al. 2011). Transfer
of viral antigen specific T cells is a well-established procedure
used for the treatment of transplant associated viral infections
and rare viral-related malignancies. Similarly, isolation and
transfer of tumor specific T cells has been shown to be successful
in treating melanoma.
[0003] Novel specificities in T cells have been successfully
generated through the genetic transfer of transgenic T cell
receptors or chimeric antigen receptors (CARS) (Jena, Dotti et al.
2010). CARs are synthetic receptors consisting of a targeting
moiety that is associated with one or more signaling domains in a
single fusion molecule. In general, the binding moiety of a CAR
consists of an antigen-binding domain of a single-chain antibody
(scFv), comprising the light and variable fragments of a monoclonal
antibody joined by a flexible linker. Binding moieties based on
receptor or ligand domains have also been used successfully. The
signaling domains for first generation CARs are derived from the
cytoplasmic region of the CD3zeta or the Fc receptor gamma chains.
First generation CARs have been shown to successfully redirect
T-cell cytotoxicity. However, they failed to provide prolonged
expansion and anti-tumor activity in vivo. Signaling domains from
co-stimulatory molecules, as well as transmembrane and hinge
domains have been added to form CARs of second and third
generations, leading to some successful therapeutic trials in
humans, where T-cells could be redirected against malignant cells
expressing CD19 (June et al., 2011). However, the particular
combination of signaling domains, transmembrane and co-stimulatory
domains used with respect to CD19 ScFv, was rather antigen-specific
and cannot be expanded to any antigen markers.
[0004] According to the data from the Centers for Disease Control
and Prevention.
[http://www.cdc.gov/cancer/colorectal/statistics/race.htm],
incidence of colorectal cancer in the US population over the year
2011 was about 50 per 100 000 people for women and up to 60 for
males in the black people population, leading to 50% mortality.
This incidence has only decreased by 10% over the last decade.
[0005] One candidate antigen of immunotherapies for solid tumors,
including the colorectal, ovarian and gastric and also for
non-solid tumors such as childhood acute lymphoblastic leukemia
(ALL) is the trophoblast glycoprotein, also known as TPBG or 5T4
(UniProt: 013641). 5T4 is often referred to as an oncofetal antigen
due to its expression in foetal trophoblast (where it was first
discovered) or trophoblast glycoprotein (TPBG). 5T4 protein is an
N-glycosylated transmembrane 72 kDa glycoprotein containing seven
leucine-rich repeat regions (Hole et al, 1988). The 5T4 antigen was
found to be expressed in number of carcinoma including gastric
(Starzynska et al. 1995), ovarian and carcinoma (Wrigley et al.
1995). Also, 5T4 oncofetal antigen is expressed in high risk of
relapse childhood pre-B acute lymphoblastic leukemia (Castro et al.
2012). It has very limited expression in normal tissue but is
widespread in malignant tumors throughout their development (Cars
berg et al. 1995).
[0006] The present inventors have thus considered that 5T4 could be
a valuable target antigen for treating solid tumors such as
colorectal, ovarian and gastric tumors, by using CAR-expressing T
cells.
[0007] As an alternative to the previous strategies, the present
invention provides with 5T4 specific CARs, which can be expressed
in immune cells to target 5T4 malignant cells with significant
clinical advantage.
[0008] There is still the need for the improvement of CAR
functionality by designing CAR architecture and using suitable
components since these parameters play a role important and a fine
tuning may be necessary.
[0009] The inventors have found that, by combining CAR architecture
to the choice of suitable components, they could obtain specific
5T4 single chain CARs with high cytotoxicity towards cancerous
target cells.
SUMMARY OF THE INVENTION
[0010] The inventors have generated 5T4 specific CAR having
different structure and comprising different scFV derived from
different 5T4 specific antibodies.
[0011] In the framework of the present invention, they have
designed and implemented at 5T4 specific CAR having one of the
polypeptide structure selected from V1 to V6 as illustrated in FIG.
2, said structure comprising an extra cellular ligand
binding-domain comprising VH and VL from a monoclonal anti-5T4
antibody, a hinge, a transmembrane domain and a cytoplasmic domain
including a CD3 zeta signaling domain and a co-stimulatory domain
from 4-1BB. Preferred CAR polypeptides of the invention comprise an
amino acid sequence selected from SEQ ID NO.19 to 42. Following
non-specific activation in vitro (e.g. with anti CD3/CD28 coated
beads and recombinant IL2), T-cells from donors have been
transformed with polynucleotides expressing these CARs using viral
transduction. More preferred CAR polypeptides having a polypeptide
structure selected from V3, V5, V1 (i.e. having the CD8a
transmembrane domain) have shown the best and unexpected
results.
[0012] In particular, the 5T4 specific CARs containing the scFvs
from A1, A2, A3 and H8 antibodies represent suitable candidates for
immunotherapy as shown by their activity and specificity tested
against selected tumor cell lines expressing the 5T4 antigen.
[0013] In certain instances, the T-cells were further engineered to
create non-alloreactive T-cells, more especially by disruption of a
component of TCR (.alpha..beta.-T-Cell receptors) to prevent Graft
versus host reaction.
[0014] The resulting engineered T-cells displayed reactivity
in-vitro against 5T4 positive cells to various extend, showing that
the CARs of the present invention contribute to antigen dependent
activation, and also proliferation, of the T-cells, making them
useful for immunotherapy.
[0015] The polypeptides and polynucleotide sequences encoding the
CARs of the present invention are detailed in the present
specification.
[0016] The engineered immune cells of the present invention are
particularly useful for therapeutic applications, such as for
treating chronic lymphocytic leukemia or on solid tumors such as
breast, colon, lung, and kidney tumors.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1: Schematic representation of an engineered immune
cell according to the invention. The engineered immune cell
presented in this figure is a T-cell transduced with a retroviral
polypeptide encoding CAR. This T-cell is further engineered to
allow a better and safer engraftment into the patient, which is
optional within the frame of the present invention. X gene may be
for instance a gene expressing a component of TCR (TCRalpha or
TCRbeta), Y may be a gene involved into the sensitivity of T-cells
to immune-suppressive drugs like CD52 (with respect to Campath) or
HPRT (with respect to 6-Thioguanine).
[0018] FIG. 2: schematic representation of the different CAR
Architecture (V1 to V6).
[0019] FIGS. 3 to 6: schematic representation of the v1 to v6 T
cell CARs accordingly to FIG. 2 with the VH and VL chains from A1,
A2, A3 and H8 antibodies.
[0020] FIG. 7: T cell degranulation test for eight
5T4-CAR-engineered T cells lines according to the invention to
assess their activity.
[0021] FIG. 8: T cell specific lysis for seven 5T4-CAR-enginereed T
cells lines according to the invention to assess their
specificity.
TABLE-US-00001 [0022] TABLE 1 Sequence of the different CAR
components Functional domains SEQ ID # Raw amino acid sequence
CD8.alpha. signal peptide SEQ ID NO. 1 MALPVTALLLPLALLLHAARP
Alternative signal peptide SEQ ID NO. 2 METDTLLLWVLLLWVPGSTG
Fc.epsilon.RIII.gamma. hinge SEQ ID NO. 3 GLAVSTISSFFPPGYQ
CD8.alpha. hinge SEQ ID NO. 4
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACD IgG1 hinge SEQ ID
NO. 5 EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIART
PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CD8.alpha. transmembrane SEQ ID
NO. 6 IYIWAPLAGTCGVLLLSLVITLYC domain 41BB transmembrane SEQ ID NO.
7 IISFFLALTSTALLFLLFFLTLRFSVV domain 41BB intracellular domain SEQ
ID NO. 8 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CEL
CD3.zeta.ntracellular domain SEQ ID NO. 9
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR
RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG
MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR G4Sx3 linker SEQ ID NO. 10
GGGGSGGGGSGGGGS
TABLE-US-00002 TABLE 2 Sequence of the VH and VL chaines of
different scFvs and their respective CDRs ScFv sequences SEQ ID #
Raw amino acid sequence H8 heavy chain SEQ ID NO. 11
EVQLQQSGPDLVKPGASVKISCKASGYSFTGYYMHWVKQSHGKSLEWIGRINP variable
region NNGVTLYNQKFKDKAILTVDKSSTTAYMELRSLTSEDSAVYYCARSTMITNYVM
DYWGQVTSVTVSS SEQ ID NO. 48 CDR1 GYSFTGYY SEQ ID NO. 49 CDR2
INPNNGVT SEQ ID NO. 50 CDR3 ARSTMITNYVMDY H8 light chain SEQ ID NO.
12 SIVMTQTPTFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPTLLISYTSSRY variable
region AGVPDRFIGSGYGTDFTFTISTLQAEDLAVYFCQQDYNSPPTFGGGTKLEIKR SEQ ID
NO. 51 CDR1 QSVSND SEQ ID NO. 52 CDR2 YTS SEQ ID NO. 53 CDR3
QQDYNSPPT A1 heavy chain SEQ ID NO. 13
QIQLVQSGPELKKPGETVKISCKASGYTFTNFGMNWVKQGPGEGLKWMGWIN variable
region TNTGEPRYAEEFKGRFAFSLETTASTAYLQINNLKNEDTATYFCARDWDGAYFFD
YWGQGTTLTVSS SEQ ID NO. 54 CDR1 GYTFTNFG SEQ ID NO. 55 CDR2
INTNTGEP SEQ ID NO. 56 CDR3 ARDWDGAYFFDY A1 light chain SEQ ID NO.
14 SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLINFATNR variable
region YTGVPNRFTGSGYGTDFTFTISTVQAEDLALYFCQQDYSSPWTFGGGTKLEIK SEQ ID
NO. 57 CDR1 QSVSND SEQ ID NO. 58 CDR2 FAT SEQ ID NO. 59 CDR3
QQDYSSPWT A2 heavy chain SEQ ID NO. 15
QVQLQQSRPELVKPGASVKMSCKASGYTFTDYVISWVKQRTGQGLEWIGEIYP variable
region GSNSIYYNEKFKGRATLTADKSSSTAYMQLSSLTSEDSAVYFCAMGGNYGFDYW
GQGTTLTVSS SEQ ID NO. 60 CDR1 GYTFTDYV SEQ ID NO. 61 CDR2 IYPGSNSI
SEQ ID NO. 62 CDR3 AMGGNYGFDY A2 light chain SEQ ID NO. 16
QIVLTQSPAIMSASLGERVILTCTASSSVNSNYLHWYQQKPGSSPKLWIYSTSNL variable
region ASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPLTFGAGTKLELK SEQ ID
NO. 63 CDR1 SSVNSNY SEQ ID NO. 64 CDR2 STS SEQ ID NO. 65 CDR3
HQYHRSPLT A3 heavy chain SEQ ID NO. 17
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIR variable
region SKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRQWDY
DVRAMNYWGQGTSVTVSS SEQ ID NO. 66 CDR1 GFTFNTYA SEQ ID NO. 67 CDR2
IRSKSNNYAT SEQ ID NO. 68 CDR3 VRQWDYDVRAMNY A3 light chain SEQ ID
NO. 18 DIVMTQSHIFMSTSVGDRVSITCKASQDVDTAVAWYQQKPGQSPKLLIYWAST
variable region
RLTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIK SEQ ID NO.
69 CDR1 QDVDTA SEQ ID NO. 70 CDR2 WAS SEQ ID NO. 71 CDR3
QQYSSYPYT
TABLE-US-00003 TABLE 3 CAR of structure V-1 CAR Structure CAR
signal Designation peptide Fc.epsilon.RIII.gamma. V-1 (optional) VH
VL hinge CD8.alpha. TM 41BB-IC CD3 CD H8 scCAR-v1 SEQ ID SEQ ID SEQ
ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 19) NO. 1 NO. 11 NO. 12
NO. 3 NO. 6 NO. 8 NO. 9 A1 scCAR-v1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ
ID SEQ ID SEQ ID (SEQ ID NO. 25) NO. 1 NO. 13 NO. 14 NO. 3 NO. 6
NO. 8 NO. 9 A2 scCAR-v1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
SEQ ID (SEQ ID NO. 31) NO. 1 NO. 15 NO. 16 NO. 3 NO. 6 NO. 8 NO. 9
A3 scCAR-v1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ
ID NO. 37) NO. 1 NO. 17 NO. 18 NO. 3 NO. 6 NO. 8 NO. 9
TABLE-US-00004 TABLE 4 CAR of structure V-2 CAR Structure CAR
signal Designation peptide Fc.epsilon.RIII.gamma. V-2 (optional) VH
VL hinge 41BB-TM 41BB-IC CD3 CD H8 scCAR-v2 SEQ ID SEQ ID SEQ ID
SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 20) NO. 1 NO. 11 NO. 12 NO.
3 NO. 7 NO. 8 NO. 9 A1-scCAR-v2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
SEQ ID SEQ ID (SEQ ID NO. 26) NO. 1 NO. 13 NO. 14 NO. 3 NO. 7 NO. 8
NO. 9 A2 scCAR-v2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
(SEQ ID NO. 32) NO. 1 NO. 15 NO. 16 NO. 3 NO. 7 NO. 8 NO. 9 A3
scCAR-v2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID
NO. 38) NO. 1 NO. 17 NO. 18 NO. 3 NO. 7 NO. 8 NO. 9
TABLE-US-00005 TABLE 5 CAR of structure V-3 CAR Structure CAR
signal Designation peptide CD8.alpha. V-3 (optional) VH VL hinge
CD8.alpha. TM 41BB-IC CD3 CD H8 scCAR-v3 SEQ ID SEQ ID SEQ ID SEQ
ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 21) NO. 1 NO. 11 NO. 12 NO. 4
NO. 6 NO. 8 NO. 9 A1-scCAR-v3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
SEQ ID SEQ ID (SEQ ID NO. 27) NO. 1 NO. 13 NO. 14 NO. 4 NO. 6 NO. 8
NO. 9 A2 scCAR-v3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
(SEQ ID NO. 33) NO. 1 NO. 15 NO. 16 NO. 4 NO. 6 NO. 8 NO. 9 A3
scCAR-v3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID
NO. 39) NO. 1 NO. 17 NO. 18 NO. 4 NO. 6 NO. 8 NO. 9
TABLE-US-00006 TABLE 6 CAR of structure V-4 CAR Structure CAR
signal Designation peptide CD8.alpha. V-4 (optional) VH VL hinge
41BB-TM 41BB-IC CD3 CD H8 scCAR-v4 SEQ ID SEQ ID SEQ ID SEQ ID SEQ
ID SEQ ID SEQ ID (SEQ ID NO. 22) NO. 1 NO. 11 NO. 12 NO. 4 NO. 7
NO. 8 NO. 9 A1 scCAR-v4 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
SEQ ID (SEQ ID NO. 28) NO. 1 NO. 13 NO. 14 NO. 4 NO. 7 NO. 8 NO. 9
A2 scCAR-v4 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ
ID NO. 34) NO. 1 NO. 15 NO. 16 NO. 4 NO. 7 NO. 8 NO. 9 A3 scCAR-v4
SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 40)
NO. 1 NO. 17 NO. 18 NO. 4 NO. 7 NO. 8 NO. 9
TABLE-US-00007 TABLE 7 CAR of structure V-5 CAR Structure CAR
signal Designation peptide V-5 (optional) VH VL IgG1 hinge
CD8.alpha. TM 41BB-IC CD3 CD H8 scCAR-v5 SEQ ID SEQ ID SEQ ID SEQ
ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 23) NO. 1 NO. 11 NO. 12 NO. 5
NO. 6 NO. 8 NO. 9 A1 scCAR-v5 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
SEQ ID SEQ ID (SEQ ID NO. 29) NO. 1 NO. 13 NO. 14 NO. 5 NO. 6 NO. 8
NO. 9 A2 scCAR-v5 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID
(SEQ ID NO. 35) NO. 1 NO. 15 NO. 16 NO. 5 NO. 6 NO. 8 NO. 9 A3
scCAR-v5 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID
NO. 41) NO. 1 NO. 17 NO. 18 NO. 5 NO. 6 NO. 8 NO. 9
TABLE-US-00008 TABLE 8 CAR of structure V-6 CAR Structure CAR
signal Designation peptide V-6 (optional) VH VL IgG1 hinge 41BB-TM
41BB-IC CD3 CD H8 scCAR-v6 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ
ID SEQ ID (SEQ ID NO. 24) NO. 1 NO. 11 NO. 12 NO. 5 NO. 7 NO. 8 NO.
9 A1 scCAR-v6 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ
ID NO. 30) NO. 1 NO. 13 NO. 14 NO. 5 NO. 7 NO. 8 NO. 9 A2 scCAR-v6
SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 36)
NO. 1 NO. 15 NO. 16 NO. 5 NO. 7 NO. 8 NO. 9 A3 scCAR-v6 SEQ ID SEQ
ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (SEQ ID NO. 42) NO. 1 NO. 17
NO. 18 NO. 5 NO. 7 NO. 8 NO. 9
DETAILED DESCRIPTION OF THE INVENTION
[0023] Unless specifically defined herein, all technical and
scientific terms used have the same meaning as commonly understood
by a skilled artisan in the fields of gene therapy, biochemistry,
genetics, and molecular biology.
[0024] All methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, with suitable methods and materials being
described herein. All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference
in their entirety. In case of conflict, the present specification,
including definitions, will prevail. Further, the materials,
methods, and examples are illustrative only and are not intended to
be limiting, unless otherwise specified.
[0025] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature. See,
for example, Current Protocols in Molecular Biology (Frederick M.
AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA);
Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et
al, 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory
Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et
al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.
Harries & S. J. Higgins eds. 1984); Transcription And
Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of
Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987);
Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A
Practical Guide To Molecular Cloning (1984); the series, Methods In
ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press,
Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.)
and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene
Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos
eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods
In Cell And Molecular Biology (Mayer and Walker, eds., Academic
Press, London, 1987); Handbook Of Experimental Immunology, Volumes
I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating
the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1986).
[0026] 5T4 Specific Chimeric Antigen Receptors
[0027] The present invention relates to new designs of anti-5T4
chimeric antigen receptor (CAR) comprising an extracellular
ligand-binding domain, a transmembrane domain and a signaling
transducing domain.
[0028] The term "extracellular ligand-binding domain" as used
herein is defined as an oligo- or polypeptide that is capable of
binding a ligand. Preferably, the domain will be capable of
interacting with a cell surface molecule. For example, the
extracellular ligand-binding domain may be chosen to recognize a
ligand that acts as a cell surface marker on target cells
associated with a particular disease state. In a preferred
embodiment, said extracellular ligand-binding domain comprises a
single chain antibody fragment (scFv) comprising the light
(V.sub.L) and the heavy (V.sub.H) variable fragment of a target
antigen specific monoclonal anti 5T4 antibody joined by a flexible
linker.
[0029] The antigen binding domain of the 5T4 CARs of the invention
can be any domain that binds to the off-tissue antigen including
but not limited to a monoclonal antibody, a recombinant antibody, a
human antibody, a humanized antibody, and a functional fragment
thereof.
[0030] By the term "recombinant antibody" as used herein, is meant
an antibody or antibody fragment which is generated using
recombinant DNA technology, such as, for example, an antibody or
antibody fragment expressed by a bacteriophage, a yeast expression
system or a mammalian cell expression system, and more especially
by a T cell transduced with a viral vector comprising a nucleic
acid sequence encoding CDR regions of an antibody. The term should
also be construed to mean an antibody or antibody fragment which
has been generated by the synthesis of a DNA molecule encoding the
antibody or antibody fragment and which DNA molecule expresses an
antibody or antibody fragment protein, or an amino acid sequence
specifying the antibody or antibody fragment, wherein the DNA or
amino acid sequence has been obtained using recombinant or
synthetic DNA or amino acid sequence technology which is available
and well known in the art.
[0031] By the term "monoclonal antibody" as used herein, is meant
antibody produced by a laboratory-grown cell clone, either of a
hybridoma or a virus-transformed lymphocyte, that is more abundant
and uniform than natural antibody and is able to bind specifically
to a single site on ROR1 antigen. They are monospecific antibodies
that are made by identical immune cells that are all clones of a
unique parent cell, in contrast to polyclonal antibodies which are
made from several different immune cells. Monoclonal antibodies
have monovalent affinity, in that they bind to the same
epitope.
[0032] In a preferred embodiment, said extracellular ligand-binding
domain comprises a single chain antibody fragment (scFv) comprising
the light (V.sub.L) and the heavy (V.sub.H) variable fragment of a
target antigen specific monoclonal 514 antibody joined by a
flexible linker. Said V.sub.L and V.sub.H are preferably selected
from the antibodies referred to as H8, A1, A2 and A3 as indicated
in Table 2. They are preferably linked together by a flexible
linker comprising for instance the sequence SEQ ID NO.10. In other
words, said CARs preferentially comprise an extracellular
ligand-binding domain comprising a polypeptide sequence displaying
at least 90%, 95% 97% or 99% identity with an amino acid sequence
selected from the group consisting of SEQ ID NO: 11 to SEQ ID NO:
18.
[0033] According to a preferred embodiment, the 5T4 specific CAR
according to the present invention contains an extracellular
ligand-binding domain, wherein said VH and VL have at least 80%,
preferably at least 90%, more preferably at least 95%, and even
more preferably at least 99% sequence identity respectively with
SEQ ID NO:13 (A1-VH) and SEQ ID NO:14 (A1-VL).
[0034] According to another preferred embodiment, the 5T4 specific
CAR according to the present invention contains an extracellular
ligand-binding domain, wherein said VH and VL have at least 80%,
preferably at least 90%, more preferably at least 95%, and even
more preferably at least 99% sequence identity respectively with
SEQ ID NO:15 (A2-VH) and SEQ ID NO:16 (A2-VL).
[0035] According to another preferred embodiment, the 5T4 specific
CAR according to the present invention contains an extracellular
ligand-binding domain, wherein said VH and VL have at least 80%,
preferably at least 90%, more preferably at least 95%, and even
more preferably at least 99% sequence identity respectively with
SEQ ID NO:17 (A3-VH) and SEQ ID NO:18 (A3-VL).
[0036] According to another preferred embodiment, the 5T4 specific
CAR according to the present invention contains an extracellular
ligand-binding domain, wherein said VH and VL have at least 80%,
preferably at least 90%, more preferably at least 95%, and even
more preferably at least 99% sequence identity respectively with
SEQ ID NO:11 (H18-VH) and SEQ ID NO:12 (H18-VL).
[0037] The present invention discloses a 5T4 specific chimeric
antigen receptor (5T4 CAR) as above, wherein said extra cellular
ligand binding-domain comprises VH and VL chains which are
humanized.
[0038] By the term "humanized antibody" as used herein, is meant
the polypeptides include a humanized heavy chain variable region
and a humanized light chain variable region. For example, the
polypeptides may include the framework (FR) regions of the light
and heavy chain variable regions of a human antibody, while
retaining substantially the antigen-binding specificity of a
parental monoclonal antibody. The humanized heavy chain variable
region and/or the humanized light chain variable region are at
least about 87% humanized, at least about 90% humanized, at least
about 95% humanized, at least about 98% humanized, or at least
about 100% humanized, excluding the complementary-determining
regions (CDRs). The antigen-binding polypeptides molecules may be
derived from monoclonal antibody donors (e.g., mouse monoclonal
antibody donors) and may include CDRs from the monoclonal
antibodies (e.g., mouse monoclonal CDRs).
[0039] A humanized antibody can be produced using a variety of
techniques known in the art, including but not limited to,
CDR-grafting (see, e.g., European Patent No. EP 239,400;
International Publication No. WO 91/09967; and U.S. Pat. Nos.
5,225,539, 5,530,101, and 5,585,089, each of which is incorporated
herein in its entirety by reference), veneering or resurfacing
(see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan,
1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al.,
1994, Protein Engineering, 7(6):805-814; each of which is
incorporated herein by its entirety by reference), chain shuffling
(see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein
in its entirety by reference), and techniques disclosed in, e.g.,
U.S. Patent Application Publication No. US2005/0042664, U.S. Patent
Application Publication No. US2005/0048617, U.S. Pat. No.
6,407,213, U.S. Pat. No. 5,766,886, International Publication No.
WO 9317105, each of which is incorporated herein in its entirety by
reference. Often, framework residues in the framework regions will
be substituted with the corresponding residue from the CDR donor
antibody to alter, for example improve, antigen binding. These
framework substitutions are identified by methods well-known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; and which is incorporated herein by reference
in their entireties).
[0040] According to a preferred embodiment, the A 5T4 specific CAR
of the present invention comprises VH and VL chains which have at
least 80%, preferably 90%, more preferably wherein said extra
cellular ligand binding-domain comprising: [0041] a VH chain
comprising the CDRs from the mouse monoclonal antibody H8 of SEQ ID
NO. 48 (CDR1), SEQ ID NO.49 (CDR2) and SEQ ID NO.50 (CDR3), and a
VL chain comprising the CDRs from the mouse monoclonal antibody H18
of NO. 51 (CDR1), SEQ ID NO.52 (CDR2) and SEQ ID NO:53 (CDR3), or;
[0042] a VH chain comprising the CDRs from the mouse monoclonal
antibody A1 of SEQ ID NO. 54 (CDR1), SEQ ID NO.55 (CDR2) and SEQ ID
NO.56 (CDR3), and a VL chain comprising the CDRs from the mouse
monoclonal antibody A1 of NO. 57 (CDR1), SEQ ID NO.58 (CDR2) and
SEQ ID NO:59 (CDR3), or; [0043] a VH chain comprising the CDRs from
the mouse monoclonal antibody A2 of SEQ ID NO. 61 (CDR1), SEQ ID
NO.61 (CDR2) and SEQ ID NO.63 (CDR3), and a VL chain comprising the
CDRs from the mouse monoclonal antibody A2 of NO. 64 (CD1), SEQ ID
NO.65 (CD2) and SEQ ID NO:65 (CDR3), or; [0044] a VH chain
comprising the CDRs from the mouse monoclonal antibody A3 of SEQ ID
NO. 66 (CDR1), SEQ ID NO.67 (CDR2) and SEQ ID NO.68 (CDR3), and a
VL chain comprising the CDRs from the mouse monoclonal antibody A3
of NO. 69 (CDR1), SEQ ID NO.70 (CDR2) and SEQ ID NO:71 (CDR3).
[0045] Table 2 shows the sequences VH and VL chains corresponding
to the H8, A1, A2 and A3 anti-5T4 antibodies and of their
respective CDRs.
[0046] The signal transducing domain or intracellular signaling
domain of a CAR according to the present invention is responsible
for intracellular signaling following the binding of extracellular
ligand binding domain to the target resulting in the activation of
the immune cell and immune response. In other words, the signal
transducing domain is responsible for the activation of at least
one of the normal effector functions of the immune cell in which
the CAR is expressed. For example, the effector function of a T
cell can be a cytolytic activity or helper activity including the
secretion of cytokines. Thus, the term "signal transducing domain"
refers to the portion of a protein which transduces the effector
signal function signal and directs the cell to perform a
specialized function.
[0047] Preferred examples of signal transducing domain for use in a
CAR can be the cytoplasmic sequences of the T cell receptor and
co-receptors that act in concert to initiate signal transduction
following antigen receptor engagement, as well as any derivate or
variant of these sequences and any synthetic sequence that has the
same functional capability. Signal transduction domain comprises
two distinct classes of cytoplasmic signaling sequence, those that
initiate antigen-dependent primary activation, and those that act
in an antigen-independent manner to provide a secondary or
co-stimulatory signal. Primary cytoplasmic signaling sequence can
comprise signaling motifs which are known as immunoreceptor
tyrosine-based activation motifs of ITAMs. ITAMs are well defined
signaling motifs found in the intracytoplasmic tail of a variety of
receptors that serve as binding sites for syk/zap70 class tyrosine
kinases. Examples of ITAM used in the invention can include as
non-limiting examples those derived from TCRzeta, FcRgamma,
FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD5, CD22,
CD79a, CD79b and CD66d. In a preferred embodiment, the signaling
transducing domain of the CAR can comprise the CD3zeta signaling
domain which has amino acid sequence with at least 70%, preferably
at least 80%, more preferably at least 90%, 95% 97% or 99% sequence
identity with amino acid sequence selected from the group
consisting of (SEQ ID NO: 9).
[0048] In particular embodiment the signal transduction domain of
the CAR of the present invention comprises a co-stimulatory signal
molecule. A co-stimulatory molecule is a cell surface molecule
other than an antigen receptor or their ligands that is required
for an efficient immune response. "Co-stimulatory ligand" refers to
a molecule on an antigen presenting cell that specifically binds a
cognate co-stimulatory molecule on a T-cell, thereby providing a
signal which, in addition to the primary signal provided by, for
instance, binding of a TCR/CD3 complex with an MHC molecule loaded
with peptide, mediates a T cell response, including, but not
limited to, proliferation activation, differentiation and the like.
A co-stimulatory ligand can include but is not limited to CD7, B7-1
(CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible
costimulatory ligand (ICOS-L), intercellular adhesion molecule
(ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM,
lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an agonist or
antibody that binds Toll ligand receptor and a ligand that
specifically binds with B7-H3. A co-stimulatory ligand also
encompasses, inter alia, an antibody that specifically binds with a
co-stimulatory molecule present on a T cell, such as but not
limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS,
lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,
NKG2C, B7-H3, a ligand that specifically binds with CD83.
[0049] A "co-stimulatory molecule" refers to the cognate binding
partner on a T-cell that specifically binds with a co-stimulatory
ligand, thereby mediating a co-stimulatory response by the cell,
such as, but not limited to proliferation. Co-stimulatory molecules
include, but are not limited to, an MHC class I molecule, BILA and
Toll ligand receptor. Examples of costimulatory molecules include
CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS,
lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,
NKG2C, B7-H3 and a ligand that specifically binds with CD83 and the
like.
[0050] In a preferred embodiment, the signal transduction domain of
the CAR of the present invention comprises a part of co-stimulatory
signal molecule selected from the group consisting of fragment of
4-1BB (GenBank: AAA53133.) and CD28 (NP_006130.1). In particular
the signal transduction domain of the CAR of the present invention
comprises amino acid sequence which comprises at least 70%,
preferably at least 80%, more preferably at least 90%, 95% 97% or
99% sequence identity with amino acid sequence selected from the
group consisting of SEQ ID NO: 8.
[0051] A CAR according to the present invention is expressed on the
surface membrane of the cell. Thus, such CAR further comprises a
transmembrane domain. The distinguishing features of appropriate
transmembrane domains comprise the ability to be expressed at the
surface of a cell, preferably in the present invention an immune
cell, in particular lymphocyte cells or Natural killer (NK) cells,
and to interact together for directing cellular response of immune
cell against a predefined target cell. The transmembrane domain can
be derived either from a natural or from a synthetic source. The
transmembrane domain can be derived from any membrane-bound or
transmembrane protein. As non-limiting examples, the transmembrane
polypeptide can be a subunit of the T-cell receptor such as
.alpha., .beta., .gamma. or .zeta. polypeptide constituting CD3
complex, IL2 receptor p55 (.alpha. chain), p75 (.beta. chain) or
.gamma. chain, subunit chain of Fc receptors, in particular
Fc.gamma. receptor III or CD proteins. Alternatively the
transmembrane domain can be synthetic and can comprise
predominantly hydrophobic residues such as leucine and valine. In a
preferred embodiment said transmembrane domain is derived from the
human CD8 alpha chain (e.g. NP_001139345.1) The transmembrane
domain can further comprise a hinge region between said
extracellular ligand-binding domain and said transmembrane domain.
The term "hinge region" used herein generally means any oligo- or
polypeptide that functions to link the transmembrane domain to the
extracellular ligand-binding domain. In particular, hinge region
are used to provide more flexibility and accessibility for the
extracellular ligand-binding domain. A hinge region may comprise up
to 300 amino acids, preferably 10 to 100 amino acids and most
preferably 25 to 50 amino acids. Hinge region may be derived from
all or part of naturally occurring molecules, such as from all or
part of the extracellular region of CD8, CD4 or CD28, or from all
or part of an antibody constant region. Alternatively the hinge
region may be a synthetic sequence that corresponds to a naturally
occurring hinge sequence, or may be an entirely synthetic hinge
sequence. In a preferred embodiment said hinge domain comprises a
part of human CD8 alpha chain, Fc.gamma.RIII.alpha. receptor or
IgG1 respectively referred to in this specification as SEQ ID NO.
3, SEQ ID NO. 4 and SEQ ID NO.5, or hinge polypeptides which
display preferably at least 80%, more preferably at least 90%, 95%
97% or 99% sequence identity with these polypeptides.
[0052] A car according to the invention generally further comprises
a transmembrane domain (TM) more particularly selected from CD8a
and 4-1BB, showing identity with the polypeptides of SEQ ID NO. 6
or 7.
[0053] Downregulation or mutation of target antigens is commonly
observed in cancer cells, creating antigen-loss escape variants.
Thus, to offset tumor escape and render immune cell more specific
to target, the 5T4 specific CAR according to the invention can
comprise another extracellular ligand-binding domains, to
simultaneously bind different elements in target thereby augmenting
immune cell activation and function. In one embodiment, the
extracellular ligand-binding domains can be placed in tandem on the
same transmembrane polypeptide, and optionally can be separated by
a linker. In another embodiment, said different extracellular
ligand-binding domains can be placed on different transmembrane
polypeptides composing the CAR. In another embodiment, the present
invention relates to a population of CARs comprising each one
different extracellular ligand binding domains. In a particular,
the present invention relates to a method of engineering immune
cells comprising providing an immune cell and expressing at the
surface of said cell a population of CAR each one comprising
different extracellular ligand binding domains. In another
particular embodiment, the present invention relates to a method of
engineering an immune cell comprising providing an immune cell and
introducing into said cell polynucleotides encoding polypeptides
composing a population of CAR each one comprising different
extracellular ligand binding domains. By population of CARs, it is
meant at least two, three, four, five, six or more CARs each one
comprising different extracellular ligand binding domains. The
different extracellular ligand binding domains according to the
present invention can preferably simultaneously bind different
elements in target thereby augmenting immune cell activation and
function. The present invention also relates to an isolated immune
cell which comprises a population of CARs each one comprising
different extracellular ligand binding domains.
[0054] According to a preferred embodiment, the 5T4 specific CAR
according to the invention has a structure V3 as displayed in FIG.
2, thus comprising a CD8a hinge and a CD8a transmembrane
domain.
[0055] According to another preferred embodiment, the 5T4 specific
CAR according to the invention has a structure V5 as displayed in
FIG. 2, thus comprising an IgG1 hinge and a CD8a transmembrane
domain.
[0056] According to another preferred embodiment, the 5T4 specific
CAR according to the invention has a structure V1 as displayed in
FIG. 2, thus comprising a Fc.gamma.RIII.alpha. hinge and CD8a
transmembrane domain.
[0057] Polynucleotides, Vectors:
[0058] The present invention also relates to polynucleotides,
vectors encoding the above described CAR according to the
invention.
[0059] The polynucleotide may consist in an expression cassette or
expression vector (e.g. a plasmid for introduction into a bacterial
host cell, or a viral vector such as a baculovirus vector for
transfection of an insect host cell, or a plasmid or viral vector
such as a lentivirus for transfection of a mammalian host
cell).
[0060] In a particular embodiment, the different nucleic acid
sequences can be included in one polynucleotide or vector which
comprises a nucleic acid sequence encoding ribosomal skip sequence
such as a sequence encoding a 2A peptide. 2A peptides, which were
identified in the Aphthovirus subgroup of picornaviruses, causes a
ribosomal "skip" from one codon to the next without the formation
of a peptide bond between the two amino acids encoded by the codons
(see (Donnelly and Elliott 2001; Atkins, Wills et al. 2007;
Doronina, Wu et al. 2008)). By "codon" is meant three nucleotides
on an mRNA (or on the sense strand of a DNA molecule) that are
translated by a ribosome into one amino acid residue. Thus, two
polypeptides can be synthesized from a single, contiguous open
reading frame within an mRNA when the polypeptides are separated by
a 2A oligopeptide sequence that is in frame. Such ribosomal skip
mechanisms are well known in the art and are known to be used by
several vectors for the expression of several proteins encoded by a
single messenger RNA.
[0061] To direct transmembrane polypeptide into the secretory
pathway of a host cell, a secretory signal sequence (also known as
a leader sequence, prepro sequence or pre sequence) is provided in
polynucleotide sequence or vector sequence. The secretory signal
sequence is operably linked to the transmembrane nucleic acid
sequence, i.e., the two sequences are joined in the correct reading
frame and positioned to direct the newly synthesized polypeptide
into the secretory pathway of the host cell. Secretory signal
sequences are commonly positioned 5' to the nucleic acid sequence
encoding the polypeptide of interest, although certain secretory
signal sequences may be positioned elsewhere in the nucleic acid
sequence of interest (see, e.g., Welch et al., U.S. Pat. No.
5,037,743; Holland et al., U.S. Pat. No. 5,143,830). In a preferred
embodiment the signal peptide comprises the amino acid sequence SEQ
ID NO: 1 and 2.
[0062] Those skilled in the art will recognize that, in view of the
degeneracy of the genetic code, considerable sequence variation is
possible among these polynucleotide molecules. Preferably, the
nucleic acid sequences of the present invention are codon-optimized
for expression in mammalian cells, preferably for expression in
human cells. Codon-optimization refers to the exchange in a
sequence of interest of codons that are generally rare in highly
expressed genes of a given species by codons that are generally
frequent in highly expressed genes of such species, such codons
encoding the amino acids as the codons that are being
exchanged.
[0063] Methods of Engineering Immune Cells Endowed with CARs:
[0064] The present invention encompasses the method of preparing
immune cells for immunotherapy comprising introducing ex-vivo into
said immune cells the polynucleotides or vectors encoding one of
the 5T4 CAR as previously described.
[0065] In a preferred embodiment, said polynucleotides are included
in lentiviral vectors in view of being stably expressed in the
immune cells.
[0066] According to further embodiments, said method further
comprises the step of genetically modifying said cell to make them
more suitable for allogeneic transplantation.
[0067] According to a first aspect, the immune cell can be made
allogeneic, for instance, by inactivating at least one gene
expressing one or more component of T-cell receptor (TCR) as
described in WO 2013/176915, which can be combined with the
inactivation of a gene encoding or regulating HLA or .beta.2m
protein expression. Accordingly the risk of graft versus host
syndrome and graft rejection is significantly reduced.
[0068] According to another aspect, the immune cells can be further
genetically engineered to improve their resistance to
immunosuppressive drugs or chemotherapy treatments, which are used
as standard care for treating 514 positive malignant cells. For
instance, CD52 and glucocorticoid receptors (GR), which are drug
targets of Campath (alemtuzumab) and glucocorticoids treatments,
can be inactivated to make the cells resistant to these treatments
and give them a competitive advantage over patient's own T-cells
not endowed with specific 5T4 CARs. Expression of CD3 gene can also
be suppressed or reduced to confer resistance to Teplizumab, which
is another immune suppressive drug. Expression of HPRT can also be
suppressed or reduced according to the invention to confer
resistance to 6-thioguanine, a cytostatic agent commonly used in
chemotherapy especially for the treatment of acute lymphoblasic
leukemia.
[0069] According to further aspect of the invention, the immune
cells can be further manipulated to make them more active or limit
exhaustion, by inactivating genes encoding proteins that act as
"immune checkpoints" that act as regulators of T-cells activation,
such as PDCD1 or CTLA-4. Examples of genes, which expression could
be reduced or suppressed are indicated in Table 9.
TABLE-US-00009 TABLE 9 List of genes encoding immune checkpoint
proteins. Genes that can be inactivated Pathway In the pathway
Co-inhibitory CTLA4 (CD152) CTLA4, PPP2CA, PPP2CB, PTPN6, receptors
PTPN22 PDCD1 (PD-1, CD279) PDCD1 CD223 (lag3) LAG3 HAVCR2 (tim3)
HAVCR2 BTLA(cd272) BTLA CD160(by55) CD160 IgSF family TIGIT CD96
CRTAM LAIR1(cd305) LAIR1 SIGLECs SIGLEC7 SIGLEC9 CD244(2b4) CD244
Death receptors TRAIL TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3,
CASP6, CASP7 FAS FADD, FAS Cytokine signalling TGF-beta signaling
TGFBRII, TGFBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1 IL10
signalling IL10RA, IL10RB, HMOX2 IL6 signalling IL6R, IL6ST
Prevention of TCR CSK, PAG1 signalling SIT1 Induced Treg induced
Treg FOXP3 Transcription transcription factors PRDM1 (=blimp1,
heterozygotes mice factors controlling controlling exhaustion
control chronic viral infection better exhaustion than wt or
conditional KO) BATF Hypoxia mediated iNOS induced guanylated
GUCY1A2, GUCY1A3, GUCY1B2, tolerance cyclase GUCY1B3
[0070] In a preferred embodiment said method of further engineering
the immune cells involves introducing into said T cells
polynucleotides, in particular mRNAs, encoding specific
rare-cutting endonuclease to selectively inactivate the genes, as
those mentioned above, by DNA cleavage. In a more preferred
embodiment said rare-cutting endonucleases are TALE-nucleases or
Cas9 endonuclease. TAL-nucleases have so far proven higher
specificity and cleavage efficiency over the other types of
rare-cutting endonucleases, making them the endonucleases of choice
for producing of the engineered immune cells on a large scale with
a constant turn-over.
[0071] Delivery Methods
[0072] The different methods described above involve introducing
CAR into a cell. As non-limiting example, said CAR can be
introduced as transgenes encoded by one plasmid vector. Said
plasmid vector can also contain a selection marker which provides
for identification and/or selection of cells which received said
vector.
[0073] Polypeptides may be synthesized in situ in the cell as a
result of the introduction of polynucleotides encoding said
polypeptides into the cell. Alternatively, said polypeptides could
be produced outside the cell and then introduced thereto. Methods
for introducing a polynucleotide construct into cells are known in
the art and including as non-limiting examples stable
transformation methods wherein the polynucleotide construct is
integrated into the genome of the cell, transient transformation
methods wherein the polynucleotide construct is not integrated into
the genome of the cell and virus mediated methods. Said
polynucleotides may be introduced into a cell by for example,
recombinant viral vectors (e.g. retroviruses, adenoviruses),
liposome and the like. For example, transient transformation
methods include for example microinjection, electroporation or
particle bombardment. Said polynucleotides may be included in
vectors, more particularly plasmids or virus, in view of being
expressed in cells.
[0074] Engineered Immune Cells
[0075] The present invention also relates to isolated cells or cell
lines susceptible to be obtained by said method to engineer cells.
In particular said isolated cell comprises at least one CAR as
described above. In another embodiment, said isolated cell
comprises a population of CARs each one comprising different
extracellular ligand binding domains. In particular, said isolated
cell comprises exogenous polynucleotide sequence encoding CAR.
Genetically modified immune cells of the present invention are
activated and proliferate independently of antigen binding
mechanisms.
[0076] In the scope of the present invention is also encompassed an
isolated immune cell, preferably a T-cell obtained according to any
one of the methods previously described. Said immune cell refers to
a cell of hematopoietic origin functionally involved in the
initiation and/or execution of innate and/or adaptative immune
response. Said immune cell according to the present invention can
be derived from a stem cell. The stem cells can be adult stem
cells, non-human embryonic stem cells, more particularly non-human
stem cells, cord blood stem cells, progenitor cells, bone marrow
stem cells, induced pluripotent stem cells, totipotent stem cells
or hematopoietic stem cells. Representative human cells are CD34+
cells. Said isolated cell can also be a dendritic cell, killer
dendritic cell, a mast cell, a NK-cell, a B-cell or a T-cell
selected from the group consisting of inflammatory T-lymphocytes,
cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper
T-lymphocytes. In another embodiment, said cell can be derived from
the group consisting of CD4+T-lymphocytes and CD8+T-lymphocytes.
Prior to expansion and genetic modification of the cells of the
invention, a source of cells can be obtained from a subject through
a variety of non-limiting methods. Cells can be obtained from a
number of non-limiting sources, including peripheral blood
mononuclear cells, bone marrow, lymph node tissue, cord blood,
thymus tissue, tissue from a site of infection, ascites, pleural
effusion, spleen tissue, and tumors. In certain embodiments of the
present invention, any number of T cell lines available and known
to those skilled in the art, may be used. In another embodiment,
said cell can be derived from a healthy donor, from a patient
diagnosed with cancer or from a patient diagnosed with an
infection. In another embodiment, said cell is part of a mixed
population of cells which present different phenotypic
characteristics. In the scope of the present invention is also
encompassed a cell line obtained from a transformed T-cell
according to the method previously described. Modified cells
resistant to an immunosuppressive treatment and susceptible to be
obtained by the previous method are encompassed in the scope of the
present invention.
[0077] As a preferred embodiment, the present invention provides
T-cells or a population of T-cells endowed with a 5T4 CAR as
described above, that do not express functional TCR and that a
reactive towards 5T4 positive cells, for their allogeneic
transplantation into patients.
[0078] Activation and Expansion of T Cells
[0079] Whether prior to or after genetic modification of the T
cells, even if the genetically modified immune cells of the present
invention are activated and proliferate independently of antigen
binding mechanisms, the immune cells, particularly T-cells of the
present invention can be further activated and expanded generally
using methods as described, for example, in U.S. Pat. Nos.
6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466;
6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843;
5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent
Application Publication No. 20060121005. T cells can be expanded in
vitro or in vivo.
[0080] Generally, the T cells of the invention are expanded by
contact with an agent that stimulates a CD3 TCR complex and a
co-stimulatory molecule on the surface of the T cells to create an
activation signal for the T-cell. For example, chemicals such as
calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or
mitogenic lectins like phytohemagglutinin (PHA) can be used to
create an activation signal for the T-cell.
[0081] As non-limiting examples, T cell populations may be
stimulated in vitro such as by contact with an anti-CD3 antibody,
or antigen-binding fragment thereof, or an anti-CD2 antibody
immobilized on a surface, or by contact with a protein kinase C
activator (e.g., bryostatin) in conjunction with a calcium
ionophore. For co-stimulation of an accessory molecule on the
surface of the T cells, a ligand that binds the accessory molecule
is used. For example, a population of T cells can be contacted with
an anti-CD3 antibody and an anti-CD28 antibody, under conditions
appropriate for stimulating proliferation of the T cells.
Conditions appropriate for T cell culture include an appropriate
media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo
5, (Lonza)) that may contain factors necessary for proliferation
and viability, including serum (e.g., fetal bovine or human serum),
interleukin-2 (IL-2), insulin, IFN-g, 1L-4, 1L-7, GM-CSF, -10, -2,
1L-15, TGFp, and TNF- or any other additives for the growth of
cells known to the skilled artisan. Other additives for the growth
of cells include, but are not limited to, surfactant, plasmanate,
and reducing agents such as N-acetyl-cysteine and
2-mercaptoethanoi. Media can include RPMI 1640, A1M-V, DMEM, MEM,
a-MEM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, with added amino
acids, sodium pyruvate, and vitamins, either serum-free or
supplemented with an appropriate amount of serum (or plasma) or a
defined set of hormones, and/or an amount of cytokine(s) sufficient
for the growth and expansion of T cells. Antibiotics, e.g.,
penicillin and streptomycin, are included only in experimental
cultures, not in cultures of cells that are to be infused into a
subject. The target cells are maintained under conditions necessary
to support growth, for example, an appropriate temperature (e.g.,
37.degree. C.) and atmosphere (e.g., air plus 5% C02). T cells that
have been exposed to varied stimulation times may exhibit different
characteristics
[0082] In another particular embodiment, said cells can be expanded
by co-culturing with tissue or cells. Said cells can also be
expanded in vivo, for example in the subject's blood after
administrating said cell into the subject.
[0083] Therapeutic Applications
[0084] In another embodiment, isolated cell obtained by the
different methods or cell line derived from said isolated cell as
previously described can be used as a medicament. In another
embodiment, said medicament can be used for treating cancer,
particularly for the treatment of carcinoma and leukemia in a
patient in need thereof. In another embodiment, said isolated cell
according to the invention or cell line derived from said isolated
cell can be used in the manufacture of a medicament for treatment
of a cancer in a patient in need thereof.
[0085] In another aspect, the present invention relies on methods
for treating patients in need thereof, said method comprising at
least one of the following steps:
[0086] (a) providing an immune-cell obtainable by any one of the
methods previously described;
[0087] (b) Administrating said transformed immune cells to said
patient,
[0088] On one embodiment, said T cells of the invention can undergo
robust in vivo T cell expansion and can persist for an extended
amount of time.
[0089] Said treatment can be ameliorating, curative or
prophylactic. It may be either part of an autologous immunotherapy
or part of an allogenic immunotherapy treatment. By autologous, it
is meant that cells, cell line or population of cells used for
treating patients are originating from said patient or from a Human
Leucocyte Antigen (HLA) compatible donor. By allogeneic is meant
that the cells or population of cells used for treating patients
are not originating from said patient but from a donor.
[0090] Cells that can be used with the disclosed methods are
described in the previous section. Said treatment can be used to
treat patients diagnosed wherein a pre-malignant or malignant
cancer condition characterized by 5T4-expressing cells, especially
by an overabundance of 5T4-expressing cells. Such conditions are
found in solid cancers or in hematologic cancers, such as childhood
pre-B acute lymphoblastic leukemia.
[0091] Solid tumors can be gastric tumors, colorectal tumors,
prostate tumors, breast tumors, lung tumors, renal tumors or
ovarian tumors.
[0092] More specifically, such treatment may be useful for
progressive hormone refractory prostate cancer in combination or
not of drug(s) such as docetaxel or granulocyte macrophage-colony
stimulating factor (GM-CSF).
[0093] Also, the engineered T cell of the invention may be used for
treating advanced solid tumors such as non-small cell lung cancer,
renal clear cell carcinoma or pancreatic cancer, in conjunction
with other drug(s) such as interleukin-2 (IL-2), docetaxel or
pemetrexed/cisplatin.
[0094] Moreover, the engineered T cell of the invention may be used
for treating prostate cancer with or without cyclophosphamide.
[0095] Lymphoproliferative disorder can be leukemia, in particular
childhood pre-B acute lymphoblastic leukemia.
[0096] Cancers that may be treated may comprise nonsolid tumors
(such as hematological tumors, including but not limited to pre-B
ALL (pedriatic indication), adult ALL, mantle cell lymphoma,
diffuse large B-cell lymphoma and the like. Types of cancers to be
treated with the CARs of the invention include, but are not limited
leukemia or lymphoid malignancies. Adult tumors/cancers and
pediatric tumors/cancers are also included.
[0097] Also, solid tumors such as stomach, colon, and ovarian
tumors can be treated by the CARs of the invention
[0098] The treatment with the engineered immune cells according to
the invention may be in combination with one or more therapies
against cancer selected from the group of antibodies therapy,
chemotherapy, cytokines therapy, dendritic cell therapy, gene
therapy, hormone therapy, laser light therapy and radiation
therapy.
[0099] According to a preferred embodiment of the invention, said
treatment can be administrated into patients undergoing an
immunosuppressive treatment. Indeed, the present invention
preferably relies on cells or population of cells, which have been
made resistant to at least one immunosuppressive agent due to the
inactivation of a gene encoding a receptor for such
immunosuppressive agent. In this aspect, the immunosuppressive
treatment should help the selection and expansion of the T-cells
according to the invention within the patient.
[0100] The administration of the cells or population of cells
according to the present invention may be carried out in any
convenient manner, including by aerosol inhalation, injection,
ingestion, transfusion, implantation or transplantation. The
compositions described herein may be administered to a patient
subcutaneously, intradermally, intratumorally, intranodally,
intramedullary, intramuscularly, by intravenous or intralymphatic
injection, or intraperitoneally. In one embodiment, the cell
compositions of the present invention are preferably administered
by intravenous injection.
[0101] The administration of the cells or population of cells can
consist of the administration of 10.sup.4-10.sup.9 cells per kg
body weight, preferably 10.sup.5 to 10.sup.6 cells/kg body weight
including all integer values of cell numbers within those ranges.
The cells or population of cells can be administrated in one or
more doses. In another embodiment, said effective amount of cells
are administrated as a single dose. In another embodiment, said
effective amount of cells are administrated as more than one dose
over a period time. Timing of administration is within the judgment
of managing physician and depends on the clinical condition of the
patient. The cells or population of cells may be obtained from any
source, such as a blood bank or a donor. While individual needs
vary, determination of optimal ranges of effective amounts of a
given cell type for a particular disease or conditions within the
skill of the art. An effective amount means an amount which
provides a therapeutic or prophylactic benefit. The dosage
administrated will be dependent upon the age, health and weight of
the recipient, kind of concurrent treatment, if any, frequency of
treatment and the nature of the effect desired.
[0102] In another embodiment, said effective amount of cells or
composition comprising those cells are administrated parenterally.
Said administration can be an intravenous administration. Said
administration can be directly done by injection within a
tumor.
[0103] In certain embodiments of the present invention, cells are
administered to a patient in conjunction with (e.g., before,
simultaneously or following) any number of relevant treatment
modalities, including but not limited to treatment with agents such
as antiviral therapy, cidofovir and interleukin-2, Cytarabine (also
known as ARA-C) or nataliziimab treatment for MS patients or
efaliztimab treatment for psoriasis patients or other treatments
for PML patients. In further embodiments, the T cells of the
invention may be used in combination with chemotherapy, radiation,
immunosuppressive agents, such as cyclosporin, azathioprine,
methotrexate, mycophenolate, and FK506, antibodies, or other
immunoablative agents such as CAMPATH, anti-CD3 antibodies or other
antibody therapies, cytoxin, fludaribine, cyclosporin, FK506,
rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and
irradiation. These drugs inhibit either the calcium dependent
phosphatase calcineurin (cyclosporine and FK506) or inhibit the
p70S6 kinase that is important for growth factor induced signaling
(rapamycin) (Henderson, Naya et al. 1991; Liu, Albers et al. 1992;
Bierer, Hollander et al. 1993). In a further embodiment, the cell
compositions of the present invention are administered to a patient
in conjunction with (e.g., before, simultaneously or following)
bone marrow transplantation, T cell ablative therapy using either
chemotherapy agents such as, fludarabine, external-beam radiation
therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or
CAMPATH, In another embodiment, the cell compositions of the
present invention are administered following B-cell ablative
therapy such as agents that react with CD20, e.g., Rituxan. For
example, in one embodiment, subjects may undergo standard treatment
with high dose chemotherapy followed by peripheral blood stem cell
transplantation. In certain embodiments, following the transplant,
subjects receive an infusion of the expanded immune cells of the
present invention. In an additional embodiment, expanded cells are
administered before or following surgery.
Other Definitions
[0104] Amino acid residues in a polypeptide sequence are designated
herein according to the one-letter code, in which, for example, Q
means Gln or Glutamine residue, R means Arg or Arginine residue and
D means Asp or Aspartic acid residue. [0105] Amino acid
substitution means the replacement of one amino acid residue with
another, for instance the replacement of an Arginine residue with a
Glutamine residue in a peptide sequence is an amino acid
substitution. [0106] Nucleotides are designated as follows:
one-letter code is used for designating the base of a nucleoside: a
is adenine, t is thymine, c is cytosine, and g is guanine. For the
degenerated nucleotides, r represents g or a (purine nucleotides),
k represents g or t, s represents g or c, w represents a or t, m
represents a or c, y represents t or c (pyrimidine nucleotides), d
represents g, a or t, v represents g, a or c, b represents g, t or
c, h represents a, t or c, and n represents g, a, t or c. [0107]
"As used herein, "nucleic acid" or "polynucleotides" refers to
nucleotides and/or polynucleotides, such as deoxyribonucleic acid
(DNA) or ribonucleic acid (RNA), oligonucleotides, fragments
generated by the polymerase chain reaction (PCR), and fragments
generated by any of ligation, scission, endonuclease action, and
exonuclease action. Nucleic acid molecules can be composed of
monomers that are naturally-occurring nucleotides (such as DNA and
RNA), or analogs of naturally-occurring nucleotides (e.g.,
enantiomeric forms of naturally-occurring nucleotides), or a
combination of both. Modified nucleotides can have alterations in
sugar moieties and/or in pyrimidine or purine base moieties. Sugar
modifications include, for example, replacement of one or more
hydroxyl groups with halogens, alkyl groups, amines, and azido
groups, or sugars can be functionalized as ethers or esters.
Moreover, the entire sugar moiety can be replaced with sterically
and electronically similar structures, such as aza-sugars and
carbocyclic sugar analogs. Examples of modifications in a base
moiety include alkylated purines and pyrimidines, acylated purines
or pyrimidines, or other well-known heterocyclic substitutes.
Nucleic acid monomers can be linked by phosphodiester bonds or
analogs of such linkages. Nucleic acids can be either single
stranded or double stranded. [0108] By chimeric antigen receptor
(CAR) is intended molecules that combine a binding domain against a
component present on the target cell, for example an antibody-based
specificity for a desired antigen (e.g., tumor antigen) with a T
cell receptor-activating intracellular domain to generate a
chimeric protein that exhibits a specific anti-target cellular
immune activity. Generally, CAR consists of an extracellular single
chain antibody (scFvFc), fused to the intracellular signaling
domain of the T cell antigen receptor complex zeta chain
(scFvFc:.zeta.) and have the ability, when expressed in T cells, to
redirect antigen recognition based on the monoclonal antibody's
specificity. CAR may sometimes comprise multiple transmembrane
polypeptides (multi-chain CARs) as described in WO2014039523. One
example of CAR used in the present invention is a CAR directing
against 5T4 antigen and can comprise as non-limiting example the
amino acid sequences: SEQ ID NO: 19 to 42. [0109] The term
"endonuclease" refers to any wild-type or variant enzyme capable of
catalyzing the hydrolysis (cleavage) of bonds between nucleic acids
within a DNA or RNA molecule, preferably a DNA molecule.
Endonucleases do not cleave the DNA or RNA molecule irrespective of
its sequence, but recognize and cleave the DNA or RNA molecule at
specific polynucleotide sequences, further referred to as "target
sequences" or "target sites". Endonucleases can be classified as
rare-cutting endonucleases when having typically a polynucleotide
recognition site greater than 12 base pairs (bp) in length, more
preferably of 14-55 bp. Rare-cutting endonucleases significantly
increase HR by inducing DNA double-strand breaks (DSBs) at a
defined locus (Perrin, Buckle et al. 1993; Rouet, Smih et al. 1994;
Choulika, Perrin et al. 1995; Pingoud and Silva 2007). Rare-cutting
endonucleases can for example be a homing endonuclease (Paques and
Duchateau 2007), a chimeric Zinc-Finger nuclease (ZFN) resulting
from the fusion of engineered zinc-finger domains with the
catalytic domain of a restriction enzyme such as Fokl (Porteus and
Carroll 2005), a Cas9 endonuclease from CRISPR system (Gasiunas,
Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et
al. 2013; Mali, Yang et al. 2013) or a chemical endonuclease
(Eisenschmidt, Lanio et al. 2005; Arimondo, Thomas et al. 2006). In
chemical endonucleases, a chemical or peptidic cleaver is
conjugated either to a polymer of nucleic acids or to another DNA
recognizing a specific target sequence, thereby targeting the
cleavage activity to a specific sequence. Chemical endonucleases
also encompass synthetic nucleases like conjugates of
orthophenanthroline, a DNA cleaving molecule, and triplex-forming
oligonucleotides (TFOs), known to bind specific DNA sequences
(Kalish and Glazer 2005). Such chemical endonucleases are comprised
in the term "endonuclease" according to the present invention.
[0110] By a "TALE-nuclease" (TALEN) is intended a fusion protein
consisting of a nucleic acid-binding domain typically derived from
a Transcription Activator Like Effector (TALE) and one nuclease
catalytic domain to cleave a nucleic acid target sequence. The
catalytic domain is preferably a nuclease domain and more
preferably a domain having endonuclease activity, like for instance
I-TevI, ColE7, NucA and Fok-I. In a particular embodiment, the TALE
domain can be fused to a meganuclease like for instance 1-CreI and
1-OnuI or functional variant thereof. In a more preferred
embodiment, said nuclease is a monomeric TALE-Nuclease. A monomeric
TALE-Nuclease is a TALE-Nuclease that does not require dimerization
for specific recognition and cleavage, such as the fusions of
engineered TAL repeats with the catalytic domain of I-TevI
described in WO2012138927. Transcription Activator like Effector
(TALE) are proteins from the bacterial species Xanthomonas comprise
a plurality of repeated sequences, each repeat comprising
di-residues in position 12 and 13 (RVD) that are specific to each
nucleotide base of the nucleic acid targeted sequence. Binding
domains with similar modular base-per-base nucleic acid binding
properties (MBBBD) can also be derived from new modular proteins
recently discovered by the applicant in a different bacterial
species. The new modular proteins have the advantage of displaying
more sequence variability than TAL repeats. Preferably, RVDs
associated with recognition of the different nucleotides are HD for
recognizing C, NG for recognizing T, NI for recognizing A, NN for
recognizing G or A, NS for recognizing A, C, G or T, HG for
recognizing T, IG for recognizing T, NK for recognizing G, HA for
recognizing C, ND for recognizing C, HI for recognizing C, HN for
recognizing G, NA for recognizing G, SN for recognizing G or A and
YG for recognizing T, TL for recognizing A, VT for recognizing A or
G and SW for recognizing A. In another embodiment, critical amino
acids 12 and 13 can be mutated towards other amino acid residues in
order to modulate their specificity towards nucleotides A, T, C and
G and in particular to enhance this specificity. TALE-nuclease have
been already described and used to stimulate gene targeting and
gene modifications (Boch, Scholze et al. 2009; Moscou and Bogdanove
2009; Christian, Cermak et al. 2010; Li, Huang et al. 2011).
Custom-made TAL-nucleases are commercially available under the
trade name TALEN.TM. (Cellectis, 8 rue de la Croix Jarry, 75013
Paris, France).
[0111] The rare-cutting endonuclease according to the present
invention can also be a Cas9 endonuclease. Recently, a new genome
engineering tool has been developed based on the RNA-guided Cas9
nuclease (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al.
2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013) from the type
II prokaryotic CRISPR (Clustered Regularly Interspaced Short
palindromic Repeats) adaptive immune system (see for review (Sorek,
Lawrence et al. 2013)). The CRISPR Associated (Cas) system was
first discovered in bacteria and functions as a defense against
foreign DNA, either viral or plasmid. CRISPR-mediated genome
engineering first proceeds by the selection of target sequence
often flanked by a short sequence motif, referred as the
protospacer adjacent motif (PAM). Following target sequence
selection, a specific crRNA, complementary to this target sequence
is engineered. Trans-activating crRNA (tracrRNA) required in the
CRISPR type II systems paired to the crRNA and bound to the
provided Cas9 protein. Cas9 acts as a molecular anchor facilitating
the base pairing of tracRNA with cRNA (Deltcheva, Chylinski et al.
2011). In this ternary complex, the dual tracrRNA:crRNA structure
acts as guide RNA that directs the endonuclease Cas9 to the cognate
target sequence. Target recognition by the Cas9-tracrRNA:crRNA
complex is initiated by scanning the target sequence for homology
between the target sequence and the crRNA. In addition to the
target sequence-crRNA complementarity, DNA targeting requires the
presence of a short motif adjacent to the protospacer (protospacer
adjacent motif--PAM). Following pairing between the dual-RNA and
the target sequence, Cas9 subsequently introduces a blunt double
strand break 3 bases upstream of the PAM motif (Garneau, Dupuis et
al. 2010).
[0112] Rare-cutting endonuclease can be a homing endonuclease, also
known under the name of meganuclease. Such homing endonucleases are
well-known to the art (Stoddard 2005). Homing endonucleases
recognize a DNA target sequence and generate a single- or
double-strand break. Homing endonucleases are highly specific,
recognizing DNA target sites ranging from 12 to 45 base pairs (bp)
in length, usually ranging from 14 to 40 bp in length. The homing
endonuclease according to the invention may for example correspond
to a LAGLIDADG endonuclease, to a HNH endonuclease, or to a GIY-YIG
endonuclease. Preferred homing endonuclease according to the
present invention can be an I-CreI variant. [0113] By "delivery
vector" or "delivery vectors" is intended any delivery vector which
can be used in the present invention to put into cell contact (i.e
"contacting") or deliver inside cells or subcellular compartments
(i.e "introducing") agents/chemicals and molecules (proteins or
nucleic acids) needed in the present invention. It includes, but is
not limited to liposomal delivery vectors, viral delivery vectors,
drug delivery vectors, chemical carriers, polymeric carriers,
lipoplexes, polyplexes, dendrimers, microbubbles (ultrasound
contrast agents), nanoparticles, emulsions or other appropriate
transfer vectors. These delivery vectors allow delivery of
molecules, chemicals, macromolecules (genes, proteins), or other
vectors such as plasmids, peptides developed by Diatos. In these
cases, delivery vectors are molecule carriers. By "delivery vector"
or "delivery vectors" is also intended delivery methods to perform
transfection. [0114] The terms "vector" or "vectors" refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. A "vector" in the present invention
includes, but is not limited to, a viral vector, a plasmid, a RNA
vector or a linear or circular DNA or RNA molecule which may
consists of a chromosomal, non-chromosomal, semi-synthetic or
synthetic nucleic acids. Preferred vectors are those capable of
autonomous replication (episomal vector) and/or expression of
nucleic acids to which they are linked (expression vectors). Large
numbers of suitable vectors are known to those of skill in the art
and commercially available.
[0115] Viral vectors include retrovirus, adenovirus, parvovirus (e.
g. adenoassociated viruses), coronavirus, negative strand RNA
viruses such as orthomyxovirus (e. g., influenza virus),
rhabdovirus (e. g., rabies and vesicular stomatitis virus),
paramyxovirus (e. g. measles and Sendai), positive strand RNA
viruses such as picornavirus and alphavirus, and double-stranded
DNA viruses including adenovirus, herpesvirus (e. g., Herpes
Simplex virus types 1 and 2, Epstein-Barr virus, cytomega-lovirus),
and poxvirus (e. g., vaccinia, fowlpox and canarypox). Other
viruses include Norwalk virus, togavirus, flavivirus, reoviruses,
papovavirus, hepadnavirus, and hepatitis virus, for example.
Examples of retroviruses include: avian leukosis-sarcoma, mammalian
C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus,
spumavirus (Coffin, J. M., Retroviridae: The viruses and their
replication, In Fundamental Virology, Third Edition, B. N. Fields,
et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
[0116] By "lentiviral vector" is meant HIV-Based lentiviral vectors
that are very promising for gene delivery because of their
relatively large packaging capacity, reduced immunogenicity and
their ability to stably transduce with high efficiency a large
range of different cell types. Lentiviral vectors are usually
generated following transient transfection of three (packaging,
envelope and transfer) or more plasmids into producer cells. Like
HIV, lentiviral vectors enter the target cell through the
interaction of viral surface glycoproteins with receptors on the
cell surface. On entry, the viral RNA undergoes reverse
transcription, which is mediated by the viral reverse transcriptase
complex. The product of reverse transcription is a double-stranded
linear viral DNA, which is the substrate for viral integration in
the DNA of infected cells. By "integrative lentiviral vectors (or
LV)", is meant such vectors as nonlimiting example, that are able
to integrate the genome of a target cell. At the opposite by
"non-integrative lentiviral vectors (or NILV)" is meant efficient
gene delivery vectors that do not integrate the genome of a target
cell through the action of the virus integrase. [0117] Delivery
vectors and vectors can be associated or combined with any cellular
permeabilization techniques such as sonoporation or electroporation
or derivatives of these techniques. [0118] By cell or cells is
intended any eukaryotic living cells, primary cells and cell lines
derived from these organisms for in vitro cultures. [0119] By
"primary cell" or "primary cells" are intended cells taken directly
from living tissue (i.e. biopsy material) and established for
growth in vitro, that have undergone very few population doublings
and are therefore more representative of the main functional
components and characteristics of tissues from which they are
derived from, in comparison to continuous tumorigenic or
artificially immortalized cell lines.
[0120] As non-limiting examples cell lines can be selected from the
group consisting of CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS
cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44
cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat
cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7
cells; Huvec cells; Molt 4 cells.
[0121] All these cell lines can be modified by the method of the
present invention to provide cell line models to produce, express,
quantify, detect, study a gene or a protein of interest; these
models can also be used to screen biologically active molecules of
interest in research and production and various fields such as
chemical, biofuels, therapeutics and agronomy as non-limiting
examples. [0122] by "mutation" is intended the substitution,
deletion, insertion of up to one, two, three, four, five, six,
seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,
fifteen, twenty, twenty five, thirty, fourty, fifty, or more
nucleotides/amino acids in a polynucleotide (cDNA, gene) or a
polypeptide sequence. The mutation can affect the coding sequence
of a gene or its regulatory sequence. It may also affect the
structure of the genomic sequence or the structure/stability of the
encoded mRNA. [0123] by "variant(s)", it is intended a repeat
variant, a variant, a DNA binding variant, a TALE-nuclease variant,
a polypeptide variant obtained by mutation or replacement of at
least one residue in the amino acid sequence of the parent
molecule. [0124] by "functional variant" is intended a
catalytically active mutant of a protein or a protein domain; such
mutant may have the same activity compared to its parent protein or
protein domain or additional properties, or higher or lower
activity. [0125] "identity" refers to sequence identity between two
nucleic acid molecules or polypeptides. Identity can be determined
by comparing a position in each sequence which may be aligned for
purposes of comparison. When a position in the compared sequence is
occupied by the same base, then the molecules are identical at that
position. A degree of similarity or identity between nucleic acid
or amino acid sequences is a function of the number of identical or
matching nucleotides at positions shared by the nucleic acid
sequences. Various alignment algorithms and/or programs may be used
to calculate the identity between two sequences, including FASTA,
or BLAST which are available as a part of the GCG sequence analysis
package (University of Wisconsin, Madison, Wis.), and can be used
with, e.g., default setting. For example, polypeptides having at
least 70%, 85%, 90%, 95%, 98% or 99% identity to specific
polypeptides described herein and preferably exhibiting
substantially the same functions, as well as polynucleotide
encoding such polypeptides, are contemplated. Unless otherwise
indicated a similarity score will be based on use of BLOSUM62. When
BLASTP is used, the percent similarity is based on the BLASTP
positives score and the percent sequence identity is based on the
BLASTP identities score. BLASTP "Identities" shows the number and
fraction of total residues in the high scoring sequence pairs which
are identical; and BLASTP "Positives" shows the number and fraction
of residues for which the alignment scores have positive values and
which are similar to each other. Amino acid sequences having these
degrees of identity or similarity or any intermediate degree of
identity of similarity to the amino acid sequences disclosed herein
are contemplated and encompassed by this disclosure. The
polynucleotide sequences of similar polypeptides are deduced using
the genetic code and may be obtained by conventional means, in
particular by reverse translating its amino acid sequence using the
genetic code. [0126] "signal-transducing domain" or "co-stimulatory
ligand" refers to a molecule on an antigen presenting cell that
specifically binds a cognate co-stimulatory molecule on a T-cell,
thereby providing a signal which, in addition to the primary signal
provided by, for instance, binding of a TCR/CD3 complex with an MHC
molecule loaded with peptide, mediates a T cell response,
including, but not limited to, proliferation activation,
differentiation and the like. A co-stimulatory ligand can include
but is not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2,
4-1BBL, OX40L, inducible costimulatory igand (ICOS-L),
intercellular adhesion molecule (ICAM, CD30L, CD40, CD70, CD83,
HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3,
ILT4, an agonist or antibody that binds Toll ligand receptor and a
ligand that specifically binds with B7-H3. A co-stimulatory ligand
also encompasses, inter alia, an antibody that specifically binds
with a co-stimulatory molecule present on a T cell, such as but not
limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS,
lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT,
NKG2C, B7-H3, a ligand that specifically binds with CD83.
[0127] A "co-stimulatory molecule" refers to the cognate binding
partner on a Tcell that specifically binds with a co-stimulatory
ligand, thereby mediating a co-stimulatory response by the cell,
such as, but not limited to proliferation. Co-stimulatory molecules
include, but are not limited to an MHC class I molecule, BTLA and
Toll ligand receptor.
[0128] A "co-stimulatory signal" as used herein refers to a signal,
which in combination with primary signal, such as TCR/CD3 ligation,
leads to T cell proliferation and/or upregulation or downregulation
of key molecules.
[0129] The term "extracellular ligand-binding domain" as used
herein is defined as an oligo- or polypeptide that is capable of
binding a ligand. Preferably, the domain will be capable of
interacting with a cell surface molecule. For example, the
extracellular ligand-binding domain may be chosen to recognize a
ligand that acts as a cell surface marker on target cells
associated with a particular disease state. Thus examples of cell
surface markers that may act as ligands include those associated
with viral, bacterial and parasitic infections, autoimmune disease
and cancer cells.
[0130] The term "subject" or "patient" as used herein includes all
members of the animal kingdom including non-human primates and
humans.
[0131] The above written description of the invention provides a
manner and process of making and using it such that any person
skilled in this art is enabled to make and use the same, this
enablement being provided in particular for the subject matter of
the appended claims, which make up a part of the original
description.
[0132] Where a numerical limit or range is stated herein, the
endpoints are included. Also, all values and subranges within a
numerical limit or range are specifically included as if explicitly
written out.
[0133] The above description is presented to enable a person
skilled in the art to make and use the invention, and is provided
in the context of a particular application and its requirements.
Various modifications to the preferred embodiments will be readily
apparent to those skilled in the art, and the generic principles
defined herein may be applied to other embodiments and applications
without departing from the spirit and scope of the invention. Thus,
this invention is not intended to be limited to the embodiments
shown, but is to be accorded the widest scope consistent with the
principles and features disclosed herein.
[0134] Having generally described this invention, a further
understanding can be obtained by reference to certain specific
examples, which are provided herein for purposes of illustration
only, and are not intended to be limiting unless otherwise
specified.
Examples
[0135] Materials and Methods
[0136] Primary Cells
[0137] Peripheral blood mononuclear cells were isolated by density
gradient centrifugation from buffy coats from healthy volunteer
donors (Etablissement Francais du Sang). T lymphocytes were then
purified using the EasySep human T cell enrichment kit (Stemcell
Technologies), and activated with Dynabeads Human T-Activator
CD3/CD28 (Life Technologies) in X-vivo 15 medium (Lonza)
supplemented with 20 ng/ml IL-2 (Miltenyi) and 5% human AB serum
(Seralab).
[0138] Cell Lines
[0139] The HCT116, MCF-7, SK-MEL-28 and Daudi cell lines were
obtained from the American Type Culture Collection. HCT116 cells
were cultured in McCoy supplemented with 10% heat-inactivated FCS,
2 mmol/L L-glutamine and 100 units/ml penicillin, and 100 .mu.g/mL
streptomycin. MCF-7 cells were cultured in DMEM supplemented with
10% heat-inactivated FCS, 2 mmol/L L-glutamine and 100 units/ml
penicillin, and 100 .mu.g/mL streptomycin and 0.01 mg/ml human
insulin. SK-MEL-28 cells were cultured in MEM supplemented with 10%
heat-inactivated FCS, 2 mmol/L L-glutamine and 100 units/ml
penicillin, and 100 .mu.g/mL streptomycin. Daudi cells were
cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2
mmol/L L-glutamine and 100 units/ml penicillin, and 100 .mu.g/mL
streptomycin.
[0140] Synthesis and Cloning of scCARs Coding Sequences
[0141] The DNA sequences encoding the scCARs were synthesized by
GenScript and cloned in a plasmid containing the T7 promoter for
the in vitro synthesis of CAR mRNA.
[0142] In Vitro Synthesis of CAR mRNA
[0143] mRNA encoding the scCARs were synthesized using as templates
linearized plasmids in which the sequence encoding the CARs is
under the control of the T7 promoter. In vitro transcription and
polyadenylation were done using the mMessage mMachine T7 Ultra kit
(Life technologies) according to the manufacturer's instructions.
RNAs were purified with RNeasy columns (Qiagen), eluted in
cytoporation medium T (Harvard Apparatus), and quantified by
measuring absorbance at 260 nm using a Nanodrop ND-1000
spectrophotometer. Quality of the RNA was verified on a denaturing
formaldehyde/MOPS agarose gel.
[0144] RNA Electroporation of T Cells
[0145] After a period of 11-12 days of activation, T lymphocytes
were transfected by electrotransfer of messenger RNA using an
AgilePulse MAX system (Harvard Apparatus). Following removal of
activation beads, cells were pelleted, resuspended in cytoporation
medium T at 25.times.10.sup.6 cells/ml. 5.times.10.sup.6 cells were
mixed with 15 .mu.g of the mRNA encoding the scCAR into a 0.4 cm
cuvette. The electroporation consisted of two 0.1 ms pulses at 1200
V followed by four 0.2 ms pulses at 130V. Following
electroporation, cells were diluted into culture medium and
incubated at 37.degree. C./5% CO.sub.2.
[0146] Degranulation Assay
[0147] A batch of 5.times.10.sup.4 T cells were co-cultured with
5.times.10.sup.4 5 T4-positive (MCF7 or HCT116) or -negative cells
(Daudi) in 0.1 ml per well in a 96-well plate. APC-labeled
anti-CD107a (BD Biosciences) was added at the beginning of the
co-culture in addition to 1 .mu.g/ml of anti-CD49d (BD
Biosciences), 1 .mu.g/ml of anti-CD28 (Miltenyi), and 1.times.
Monensin solution (eBioscience). After a 6 h incubation, the cells
were stained with a fixable viability dye (eBioscience) and
vioblue-labeled anti-CD8 (Miltenyi) and analyzed using the
MACSQuant flow cytometer (Miltenyi). Degranulating cytotoxic T
cells correspond to CD8+CD107a+ cells.
[0148] Cytotoxicity Assay
[0149] 5T4-positive and -negative cells were respectively labeled
with CellTrace CFSE and CellTrace Violet. Un batch of
2.times.10.sup.4 5 T4-positive cells (MCF7 or HCT116) were
co-cultured with 2.times.10.sup.4 5 T4-negative cells (SKMEL28)
with 4.times.10.sup.5 T cells in 0.1 ml per well in a 96-well
plate. After a 4 hours incubation, the cells were harvested and
stained with a fixable viability dye (eBioscience) and analyzed
using the MACSQuant flow cytometer (Miltenyi).
[0150] The percentage of specific lysis was calculated using the
following formula:
% cell lysis = 100 % - % viable target cells upon coculture with
CAR modified T cells % viable control cells upon coculture with CAR
modified T cells % viable target cells upon coculture with non
modified T cells % viable control cells upon coculture with non
modified T cells ##EQU00001##
Example 1: Proliferation of TCRalpha Inactivated Cells Expressing a
5T4-CAR
[0151] Heterodimeric TALE-nuclease targeting two 17-bp long
sequences (called half targets) separated by an 15-bp spacer within
T-cell receptor alpha constant chain region (TRAC) gene were
designed and produced. Each half target is recognized by repeats of
the half TALE-nucleases listed in Table 10
TABLE-US-00010 TABLE 10 TAL-nucleases targeting TCRalpha gene
Polynucleotid Target Target sequence encode TALEN Half
TALE-nuclease TRAC_T01 TTGTCCCACAGATATCC T01-L TRAC_T01-L TALEN
Agaaccctgaccctg (SEQ ID NO: 44) (SEQ ID NO: 46) CCGTGTACCAGCTGAGA
T01-R TRAC_T01-R TALEN (SEQ ID NO: 43) (SEQ ID NO: 45) (SEQ ID NO:
47)
[0152] Each TALE-nuclease construct was subcloned using restriction
enzyme digestion in a mammalian expression vector under the control
of the T7 promoter. mRNA encoding TALE-nuclease cleaving TRAC
genomic sequence were synthesized from plasmid carrying the coding
sequence downstream from the T7 promoter.
[0153] Purified T cells preactivated during 72 hours with
anti-CD3/CD28 coated beads were transfected with each of the 2
mRNAs encoding both half TRAC_T01 TALE-nucleases. 48 hours
post-transfection, different groups of T cells from the same donor
were respectively transduced with a lentiviral vector encoding one
of the 5T4 CAR previously described (SEQ ID NO: 19 to 42). 2 days
post-transduction, CD3.sub.NEG cells were purified using anti-CD3
magnetic beads and 5 days post-transduction cells were reactivated
with soluble anti-CD28 (5 .mu.g/ml).
[0154] Cell proliferation was followed for up to 30 days after
reactivation by counting cell 2 times per week. Increased
proliferation in TCR alpha inactivated cells expressing the 5T4
CARs, especially when reactivated with anti-CD28, was observed
compared to non-transduced cells.
[0155] To investigate whether the human T cells expressing the 5T4
CAR display activated state, the expression of the activation
marker CD25 are analyzed by FACS 7 days post transduction. The
purified cells transduced with the lentiviral vector encoding 5T4
CAR assayed for CD25 expression at their surface in order to assess
their activation in comparison with the non-transduced cells.
Increased CD25 expression is expected both in CD28 reactivation or
no reactivation conditions.
Example 2: Selection of 5T4-Positive and -Negative Cell Line
[0156] Eight human cell lines were screened for 5T4 expression by
western blot and flow cytometry (see Table 11 below).
TABLE-US-00011 TABLE 11 Expression of 5T4 antigen in 8 human cell
lines Cell line Description Cell type MCF7 adherent adenocarcinoma
HCT116 adherent colorectal carcinoma MKN45 adherent gastric
carcinoma LS174T adherent colorectal adecarcinoma SK-MEL-28
adherent malignant melanoma SupT1 suspension T-cell lymphoblastic
lymphoma Daudi suspension Burkitt's lymphoma
[0157] 5T4 was not detected in extracts from Daudi (ATCC CCL-213),
SupT1 (ATT CRL-1942) and SK-MEL-28 (ATCC HTB-72) cells but was
detected in extracts from MCF7 (ATCC HTB-22), HCT116 (ATCC
CCL-247), MKN45 (JCRB0254) and LS174T (ATCC CL-188) cells. Among
the cells that were positive for 5T4 antigen following western blot
analysis, only two were found to express 5T4 at the cell surface:
MCF7 and HCT116 cells, MCF7 expressing highest levels of 5T4
antigen than HCT116 cells.
Example 3: Generation of Anti-5T4 scCARs
[0158] Second generation singlechain CARs specific for 5T4 (shown
schematically in FIGS. 3 to 6 and and in Table 3 to Table 6) were
created by combining the sequences of 4 different scFv with the
sequences of 3 different spacers, 2 different transmembrane
domains, 1 costimulatory domain and 1 stimulatory domain as
represented in FIG. 2.
[0159] The sequences used in the CARs (presented in Table 1 and
Table 2) derive from: [0160] the H8, A1, A2 or A3 antibodies for
the scFv; [0161] the IgG1, Fc.epsilon.RIII.gamma. or CD8.alpha.
molecules for the spacer domain; [0162] the CD8.alpha. or 4-1BB
molecules for the transmembrane domain; [0163] the 4-1BB molecule
for the costimulatory domain; [0164] the CD3.zeta. molecule for the
stimulatory domain.
Example 4: In Vitro Testing of Anti-5T4 scCARs
[0165] To evaluate the activity of 5T4-specific singlechain CARs,
human T cells from healthy volunteers were activated with CD3/CD28
beads and, eleven days post activation, were electroporated with
mRNA encoding the CARs. CAR's activity and specificity were
analysed 1-2 days post transfection by measuring T cels
degranulation and T cell cytotoxicity against 5T4-positive and
-negative target cells.
[0166] The results are presented below for the testing on one case
(N=1), however experiments were performed on two other cases
showing similar results (not shown).
[0167] FIG. 7 shows that all the CARs tested induced significant
level (20%) of T cells degranulation upon coculture with MCF7 but
not upon coculture with Daudi cells. Among the eight CARs tested
seven were also able to mediate T cells degranulation following
coculture with HCT116 cells, a cell line expressing lower level of
5T4 than MCF7.
[0168] FIG. 8 shows that all the T cells modified with the A1-v3,
A1-v5, A2-v3, A2-v5, A3-v3, H8-v2 and H8-v3 CARs lysed
significantly and specifically MCF7 cells. T cells modified with
the A1-v3, A1-v5, A2-v3, A2-v5, A3-v3 and H8-v3 CARs were also able
to lyse HCT116 cells, a cell line expressing lower level of 5T4
than MCF7 cells.
Examples of CAR Polypeptide Sequences
[0169] Framed sequences correspond to preferred VH and VL
sequences. VH and VL may be swapped to improve CAR efficiency.
TABLE-US-00012 H8 v1 ##STR00001## ##STR00002## ##STR00003##
##STR00004##
KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKSRSADAPAYQQSQNQLYNELNLGRREEYDVLDK-
R
RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP
R H8 v2 ##STR00005## ##STR00006## ##STR00007## ##STR00008##
RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL-
DK
RRGRDPEMGGKPRRKNPQEGLYNELKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP
PR H8 v3 ##STR00009## ##STR00010## ##STR00011## ##STR00012##
ACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGSCRFPEEEEGGCELRVKFSR-
SADA
PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDMAEAYSEIGMKGERRRG
KGHDGLYQGLSTATKDTYDALHMQALPPR H8 v4 ##STR00013## ##STR00014##
##STR00015## ##STR00016##
ACDIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV-
KFSRSAD
APAYQQGQNQLYNELNLGREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR
GKGHDGLYQGLSTATKDTYDALHMQALPPR H8 v5 ##STR00017## ##STR00018##
##STR00019## ##STR00020##
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY-
SKLT
VDKSRWQQGNVSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVTTLYCKRGRKKLLYIFKQPF-
MR
PVQTTQEEDGCSCRFPEEEEGGCELRVKSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR H8
v6 ##STR00021## ##STR00022## ##STR00023## ##STR00024##
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI-
E
KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS-
KLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIF-
KQPFM
RPVQTTQEEDGCSCRFPEEEEGGCELRVKSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP
RRKNPQEGLYNELQKDMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR A1
v1 ##STR00025## ##STR00026## ##STR00027## ##STR00028##
RKKLLYIFQPFMRPVQTTQEEDGCSCRFPEEEEGGCELVFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK
RRGRDPEMGGKPRRKNPQEGLYNELQKDMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP
PR A1 v2 ##STR00029## ##STR00030## ##STR00031## ##STR00032##
GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV-
LD
KRRGRDPEMGGPRRKNPQEGLYNELQKDMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL
PPR A1 v3 ##STR00033## ##STR00034## ##STR00035## ##STR00036##
FACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKF-
SRSAD
APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR
GKGHDGLYQGLSTATKDTYDALHMQALPPR A1 v4 ##STR00037## ##STR00038##
##STR00039## ##STR00040##
FACDIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR-
VKFSRSA
DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDMAEAYSEIGMKGERR
GKGHDGLYQGLSTATKDTYDALHMQALPPR A1 v5 ##STR00041## ##STR00042##
##STR00043## ##STR00044##
EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP-
I
EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL-
YSKL
TVDKSRWQQGNVSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP-
FM
RPVQTTQEEDGCSCRFPEEEEGGCELRVKSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP
RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR A1
v6 ##STR00045## ##STR00046## ##STR00047## ##STR00048##
EVTCVVVDVSHEDPEVKFNWYDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL-
YSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIIFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIF-
KQPF
MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGS
KPRRKNPQEGLYNELQKDMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR A2
v1 ##STR00049## ##STR00050## ##STR00051## ##STR00052##
LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR-
RG
RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
A2 v2 ##STR00053## ##STR00054## ##STR00055## ##STR00056##
KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK-
RR
GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLTATKDTYDALHMQALPPR
A2 v3 ##STR00057## ##STR00058## ##STR00059## ##STR00060##
DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS-
ADAPA
YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG
HDGLYQGLSTATKDTDALHMQALPPR A2 v4 ##STR00061## ##STR00062##
##STR00063## ##STR00064##
DIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKF-
RSADAP
AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK
GHDGLYQGLSTATKDTYDALHMQALPPR A2 v5 ##STR00065## ##STR00066##
##STR00067## ##STR00068##
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK-
T
ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL-
TVC
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFQPFMR-
PV
QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR-
K NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR A2
v6 ##STR00069## ##STR00070## ##STR00071## ##STR00072##
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGEYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSGSFFLYSKL-
TVD
KSRWQQGNFSCSVMHEALHNHYTQKSLSLSPGKIISFFLATSTALLFLLFFLTRFSVVKRGRKKLLYIFKQPFM-
RP
VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
RKNPQEGLYNELQKDMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYALHMQALPPR A3 v1
##STR00073## ##STR00074## ##STR00075## ##STR00076##
ITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG-
RRE
EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDMAEAYSEIGMKGERRGKGHDGLYQGLSTATKDTYDAL
HMQALPPR A3 v2 ##STR00077## ##STR00078## ##STR00079## ##STR00080##
RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLG-
RR
EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA
LHMQALPPR A3 v3 ##STR00081## ##STR00082## ##STR00083## ##STR00084##
VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG-
CELRV
KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG
MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR A3 v4 ##STR00085##
##STR00086## ##STR00087## ##STR00088##
VHTRGLDFACDIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE-
EGGCELR
VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPAMGGKPRRKNPQEGLYNELQKDMAEAYSEIG
MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR A3 v5 ##STR00089##
##STR00090## ##STR00091## ##STR00092##
TLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV-
S
NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKFYPSDIAVEWSNGQPENNYKTTPPVLDS-
DG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKK-
LLYI
FKQFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP
EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
A3 v6 ##STR00093## ##STR00094## ##STR00095## ##STR00096##
TLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV-
S
NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL-
DSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIISFFLALTSTALLFLLFFLTLRFSVVKR-
GRKKLL
YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR
DPEMGGKPRRKNPQEGLYNELQKDMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
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Sequence CWU 1
1
71120PRTartificial sequencesignal peptide 1Met Glu Thr Asp Thr Leu
Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly
20 221PRTartificial sequencesignal peptide 2Met Ala Leu Pro Val Thr
Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg
Pro 20 316PRThomo sapiensFcgRIIIa hinge 3Gly Leu Ala Val Ser Thr
Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 1 5 10 15 445PRThomo
sapiensCD8alpha hinge 4Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro
Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu
Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly
Leu Asp Phe Ala Cys Asp 35 40 45 5231PRThomo sapiensIgG1 hinge 5Glu
Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10
15 Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
20 25 30 Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys Val
Val Val 35 40 45 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp 50 55 60 Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr 65 70 75 80 Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp 85 90 95 Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 100 105 110 Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125 Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 130 135 140
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 145
150 155 160 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 165 170 175 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 180 185 190 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser 195 200 205 Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser 210 215 220 Leu Ser Leu Ser Pro Gly
Lys 225 230 624PRThomo sapiensCD8alpha transmembrane domain 6Ile
Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10
15 Ser Leu Val Ile Thr Leu Tyr Cys 20 727PRThomo sapiens41BB
transmembrane domain 7Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr
Ala Leu Leu Phe Leu 1 5 10 15 Leu Phe Phe Leu Thr Leu Arg Phe Ser
Val Val 20 25 842PRThomo sapiensFragment of 4-1BB (residues
214-255) 8Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35
40 9112PRThomo sapiensfragment of T-cell surface glycoprotein CD3
zeta chain 9Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu
Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110
1015PRTartificial sequencelinker sequence 10Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 11120PRTartificial
sequenceH8 heavy chain 11Glu Val Gln Leu Gln Gln Ser Gly Pro Asp
Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Lys
Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asn
Pro Asn Asn Gly Val Thr Leu Tyr Asn Gln Lys Phe 50 55 60 Lys Asp
Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Ser Thr Met Ile Thr Asn Tyr Val Met Asp Tyr Trp Gly
Gln 100 105 110 Val Thr Ser Val Thr Val Ser Ser 115 120
12108PRTartificial sequenceH8 light chain variable region 12Ser Ile
Val Met Thr Gln Thr Pro Thr Phe Leu Leu Val Ser Ala Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20
25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Thr Leu Leu
Ile 35 40 45 Ser Tyr Thr Ser Ser Arg Tyr Ala Gly Val Pro Asp Arg
Phe Ile Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile
Ser Thr Leu Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln
Gln Asp Tyr Asn Ser Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys Arg 100 105 13119PRTartificial sequenceA3 heavy
chain 13Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly
Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Asn Phe 20 25 30 Gly Met Asn Trp Val Lys Gln Gly Pro Gly Glu
Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Asn Thr Gly Glu
Pro Arg Tyr Ala Glu Glu Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser
Leu Glu Thr Thr Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn
Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Asp
Trp Asp Gly Ala Tyr Phe Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr
Thr Leu Thr Val Ser Ser 115 14107PRTartificial sequenceA3 light
chain 14Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala
Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val
Ser Asn Asp 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser
Pro Lys Leu Leu Ile 35 40 45 Asn Phe Ala Thr Asn Arg Tyr Thr Gly
Val Pro Asn Arg Phe Thr Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe
Thr Phe Thr Ile Ser Thr Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu
Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Pro Trp 85 90 95 Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys 100 105 15117PRTartificial
sequenceA2 heavy chain 15Gln Val Gln Leu Gln Gln Ser Arg Pro Glu
Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Val Ile Ser Trp Val Lys
Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr
Pro Gly Ser Asn Ser Ile Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Gly
Arg Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85
90 95 Ala Met Gly Gly Asn Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr
Thr 100 105 110 Leu Thr Val Ser Ser 115 16108PRTartificial
sequenceA2 light chain 16Gln Ile Val Leu Thr Gln Ser Pro Ala Ile
Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Leu Thr Cys Thr
Ala Ser Ser Ser Val Asn Ser Asn 20 25 30 Tyr Leu His Trp Tyr Gln
Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr
Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85
90 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105
17122PRTartificial sequenceA3 heavy chain 17Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50
55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser
Met 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr
Ala Met Tyr 85 90 95 Tyr Cys Val Arg Gln Trp Asp Tyr Asp Val Arg
Ala Met Asn Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser
Ser 115 120 18107PRTartificial sequenceA3 light chain 18Asp Ile Val
Met Thr Gln Ser His Ile Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp
Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Asp Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45 Tyr Trp Ala Ser Thr Arg Leu Thr Gly Val Pro Asp Arg Phe
Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln
Tyr Ser Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 105 19458PRTartificial sequenceH8-v1 polypeptide
CAR sequence 19Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Gln Gln
Ser Gly Pro Asp Leu 20 25 30 Val Lys Pro Gly Ala Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Ser Phe Thr Gly Tyr Tyr Met
His Trp Val Lys Gln Ser His Gly Lys 50 55 60 Ser Leu Glu Trp Ile
Gly Arg Ile Asn Pro Asn Asn Gly Val Thr Leu 65 70 75 80 Tyr Asn Gln
Lys Phe Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser 85 90 95 Ser
Thr Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser 100 105
110 Ala Val Tyr Tyr Cys Ala Arg Ser Thr Met Ile Thr Asn Tyr Val Met
115 120 125 Asp Tyr Trp Gly Gln Val Thr Ser Val Thr Val Ser Ser Gly
Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Ser Ile Val Met 145 150 155 160 Thr Gln Thr Pro Thr Phe Leu Leu Val
Ser Ala Gly Asp Arg Val Thr 165 170 175 Ile Thr Cys Lys Ala Ser Gln
Ser Val Ser Asn Asp Val Ala Trp Tyr 180 185 190 Gln Gln Lys Pro Gly
Gln Ser Pro Thr Leu Leu Ile Ser Tyr Thr Ser 195 200 205 Ser Arg Tyr
Ala Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Tyr Gly 210 215 220 Thr
Asp Phe Thr Phe Thr Ile Ser Thr Leu Gln Ala Glu Asp Leu Ala 225 230
235 240 Val Tyr Phe Cys Gln Gln Asp Tyr Asn Ser Pro Pro Thr Phe Gly
Gly 245 250 255 Gly Thr Lys Leu Glu Ile Lys Arg Gly Leu Ala Val Ser
Thr Ile Ser 260 265 270 Ser Phe Phe Pro Pro Gly Tyr Gln Ile Tyr Ile
Trp Ala Pro Leu Ala 275 280 285 Gly Thr Cys Gly Val Leu Leu Leu Ser
Leu Val Ile Thr Leu Tyr Cys 290 295 300 Lys Arg Gly Arg Lys Lys Leu
Leu Tyr Ile Phe Lys Gln Pro Phe Met 305 310 315 320 Arg Pro Val Gln
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 325 330 335 Pro Glu
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 340 345 350
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 355
360 365 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
Arg 370 375 380 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
Lys Asn Pro 385 390 395 400 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
Asp Lys Met Ala Glu Ala 405 410 415 Tyr Ser Glu Ile Gly Met Lys Gly
Glu Arg Arg Arg Gly Lys Gly His 420 425 430 Asp Gly Leu Tyr Gln Gly
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 435 440 445 Ala Leu His Met
Gln Ala Leu Pro Pro Arg 450 455 20461PRTartificial sequenceH8-v2
polypeptide CAR sequence 20Met Ala Leu Pro Val Thr Ala Leu Leu Leu
Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln
Leu Gln Gln Ser Gly Pro Asp Leu 20 25 30 Val Lys Pro Gly Ala Ser
Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Ser Phe Thr Gly
Tyr Tyr Met His Trp Val Lys Gln Ser His Gly Lys 50 55 60 Ser Leu
Glu Trp Ile Gly Arg Ile Asn Pro Asn Asn Gly Val Thr Leu 65 70 75 80
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser 85
90 95 Ser Thr Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp
Ser 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Ser Thr Met Ile Thr Asn
Tyr Val Met 115 120 125 Asp Tyr Trp Gly Gln Val Thr Ser Val Thr Val
Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Ser Ile Val Met 145 150 155 160 Thr Gln Thr Pro Thr Phe
Leu Leu Val Ser Ala Gly Asp Arg Val Thr 165 170 175 Ile Thr Cys Lys
Ala Ser Gln Ser Val Ser Asn Asp Val Ala Trp Tyr 180 185 190 Gln Gln
Lys Pro Gly Gln Ser Pro Thr Leu Leu Ile Ser Tyr Thr Ser 195 200 205
Ser Arg Tyr Ala Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Tyr Gly 210
215 220 Thr Asp Phe Thr Phe Thr Ile Ser Thr Leu Gln Ala Glu Asp Leu
Ala 225 230 235 240 Val Tyr Phe Cys Gln Gln Asp Tyr Asn Ser Pro Pro
Thr Phe Gly Gly 245 250
255 Gly Thr Lys Leu Glu Ile Lys Arg Gly Leu Ala Val Ser Thr Ile Ser
260 265 270 Ser Phe Phe Pro Pro Gly Tyr Gln Ile Ile Ser Phe Phe Leu
Ala Leu 275 280 285 Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu
Thr Leu Arg Phe 290 295 300 Ser Val Val Lys Arg Gly Arg Lys Lys Leu
Leu Tyr Ile Phe Lys Gln 305 310 315 320 Pro Phe Met Arg Pro Val Gln
Thr Thr Gln Glu Glu Asp Gly Cys Ser 325 330 335 Cys Arg Phe Pro Glu
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 340 345 350 Phe Ser Arg
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 355 360 365 Leu
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 370 375
380 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
385 390 395 400 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
Asp Lys Met 405 410 415 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
Glu Arg Arg Arg Gly 420 425 430 Lys Gly His Asp Gly Leu Tyr Gln Gly
Leu Ser Thr Ala Thr Lys Asp 435 440 445 Thr Tyr Asp Ala Leu His Met
Gln Ala Leu Pro Pro Arg 450 455 460 21487PRTartificial
sequenceH8-v3 polypeptide CAR sequence 21Met Ala Leu Pro Val Thr
Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg
Pro Glu Val Gln Leu Gln Gln Ser Gly Pro Asp Leu 20 25 30 Val Lys
Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr 35 40 45
Ser Phe Thr Gly Tyr Tyr Met His Trp Val Lys Gln Ser His Gly Lys 50
55 60 Ser Leu Glu Trp Ile Gly Arg Ile Asn Pro Asn Asn Gly Val Thr
Leu 65 70 75 80 Tyr Asn Gln Lys Phe Lys Asp Lys Ala Ile Leu Thr Val
Asp Lys Ser 85 90 95 Ser Thr Thr Ala Tyr Met Glu Leu Arg Ser Leu
Thr Ser Glu Asp Ser 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Ser Thr
Met Ile Thr Asn Tyr Val Met 115 120 125 Asp Tyr Trp Gly Gln Val Thr
Ser Val Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Ser Ile Val Met 145 150 155 160 Thr Gln
Thr Pro Thr Phe Leu Leu Val Ser Ala Gly Asp Arg Val Thr 165 170 175
Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala Trp Tyr 180
185 190 Gln Gln Lys Pro Gly Gln Ser Pro Thr Leu Leu Ile Ser Tyr Thr
Ser 195 200 205 Ser Arg Tyr Ala Gly Val Pro Asp Arg Phe Ile Gly Ser
Gly Tyr Gly 210 215 220 Thr Asp Phe Thr Phe Thr Ile Ser Thr Leu Gln
Ala Glu Asp Leu Ala 225 230 235 240 Val Tyr Phe Cys Gln Gln Asp Tyr
Asn Ser Pro Pro Thr Phe Gly Gly 245 250 255 Gly Thr Lys Leu Glu Ile
Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro 260 265 270 Pro Thr Pro Ala
Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 275 280 285 Glu Ala
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305
310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys
Arg Gly 325 330 335 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
Met Arg Pro Val 340 345 350 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
Cys Arg Phe Pro Glu Glu 355 360 365 Glu Glu Gly Gly Cys Glu Leu Arg
Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala Pro Ala Tyr Gln Gln
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390 395 400 Leu Gly Arg
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415 Asp
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 420 425
430 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
435 440 445 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
Gly Leu 450 455 460 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
Asp Ala Leu His 465 470 475 480 Met Gln Ala Leu Pro Pro Arg 485
22490PRTartificial sequenceH8-v4 polypeptide CAR sequence 22Met Ala
Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Gln Gln Ser Gly Pro Asp Leu 20
25 30 Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr 35 40 45 Ser Phe Thr Gly Tyr Tyr Met His Trp Val Lys Gln Ser
His Gly Lys 50 55 60 Ser Leu Glu Trp Ile Gly Arg Ile Asn Pro Asn
Asn Gly Val Thr Leu 65 70 75 80 Tyr Asn Gln Lys Phe Lys Asp Lys Ala
Ile Leu Thr Val Asp Lys Ser 85 90 95 Ser Thr Thr Ala Tyr Met Glu
Leu Arg Ser Leu Thr Ser Glu Asp Ser 100 105 110 Ala Val Tyr Tyr Cys
Ala Arg Ser Thr Met Ile Thr Asn Tyr Val Met 115 120 125 Asp Tyr Trp
Gly Gln Val Thr Ser Val Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ile Val Met 145 150
155 160 Thr Gln Thr Pro Thr Phe Leu Leu Val Ser Ala Gly Asp Arg Val
Thr 165 170 175 Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val
Ala Trp Tyr 180 185 190 Gln Gln Lys Pro Gly Gln Ser Pro Thr Leu Leu
Ile Ser Tyr Thr Ser 195 200 205 Ser Arg Tyr Ala Gly Val Pro Asp Arg
Phe Ile Gly Ser Gly Tyr Gly 210 215 220 Thr Asp Phe Thr Phe Thr Ile
Ser Thr Leu Gln Ala Glu Asp Leu Ala 225 230 235 240 Val Tyr Phe Cys
Gln Gln Asp Tyr Asn Ser Pro Pro Thr Phe Gly Gly 245 250 255 Gly Thr
Lys Leu Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro 260 265 270
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 275
280 285 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
Leu 290 295 300 Asp Phe Ala Cys Asp Ile Ile Ser Phe Phe Leu Ala Leu
Thr Ser Thr 305 310 315 320 Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr
Leu Arg Phe Ser Val Val 325 330 335 Lys Arg Gly Arg Lys Lys Leu Leu
Tyr Ile Phe Lys Gln Pro Phe Met 340 345 350 Arg Pro Val Gln Thr Thr
Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 355 360 365 Pro Glu Glu Glu
Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 370 375 380 Ser Ala
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 385 390 395
400 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
405 410 415 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
Asn Pro 420 425 430 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
Met Ala Glu Ala 435 440 445 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
Arg Arg Gly Lys Gly His 450 455 460 Asp Gly Leu Tyr Gln Gly Leu Ser
Thr Ala Thr Lys Asp Thr Tyr Asp 465 470 475 480 Ala Leu His Met Gln
Ala Leu Pro Pro Arg 485 490 23490PRTartificial sequenceH8-v5
polypeptide CAR sequence 23Met Ala Leu Pro Val Thr Ala Leu Leu Leu
Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln
Leu Gln Gln Ser Gly Pro Asp Leu 20 25 30 Val Lys Pro Gly Ala Ser
Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Ser Phe Thr Gly
Tyr Tyr Met His Trp Val Lys Gln Ser His Gly Lys 50 55 60 Ser Leu
Glu Trp Ile Gly Arg Ile Asn Pro Asn Asn Gly Val Thr Leu 65 70 75 80
Tyr Asn Gln Lys Phe Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser 85
90 95 Ser Thr Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp
Ser 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Ser Thr Met Ile Thr Asn
Tyr Val Met 115 120 125 Asp Tyr Trp Gly Gln Val Thr Ser Val Thr Val
Ser Ser Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Ser Ile Val Met 145 150 155 160 Thr Gln Thr Pro Thr Phe
Leu Leu Val Ser Ala Gly Asp Arg Val Thr 165 170 175 Ile Thr Cys Lys
Ala Ser Gln Ser Val Ser Asn Asp Val Ala Trp Tyr 180 185 190 Gln Gln
Lys Pro Gly Gln Ser Pro Thr Leu Leu Ile Ser Tyr Thr Ser 195 200 205
Ser Arg Tyr Ala Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Tyr Gly 210
215 220 Thr Asp Phe Thr Phe Thr Ile Ser Thr Leu Gln Ala Glu Asp Leu
Ala 225 230 235 240 Val Tyr Phe Cys Gln Gln Asp Tyr Asn Ser Pro Pro
Thr Phe Gly Gly 245 250 255 Gly Thr Lys Leu Glu Ile Lys Arg Thr Thr
Thr Pro Ala Pro Arg Pro 260 265 270 Pro Thr Pro Ala Pro Thr Ile Ala
Ser Gln Pro Leu Ser Leu Arg Pro 275 280 285 Glu Ala Cys Arg Pro Ala
Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys
Asp Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr 305 310 315 320 Ala
Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val 325 330
335 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
340 345 350 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
Arg Phe 355 360 365 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
Lys Phe Ser Arg 370 375 380 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
Gln Asn Gln Leu Tyr Asn 385 390 395 400 Glu Leu Asn Leu Gly Arg Arg
Glu Glu Tyr Asp Val Leu Asp Lys Arg 405 410 415 Arg Gly Arg Asp Pro
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 420 425 430 Gln Glu Gly
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 435 440 445 Tyr
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 450 455
460 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
465 470 475 480 Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490
24676PRTartificial sequenceH8-v6 polypeptide CAR sequence 24Met Ala
Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Gln Gln Ser Gly Pro Asp Leu 20
25 30 Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr 35 40 45 Ser Phe Thr Gly Tyr Tyr Met His Trp Val Lys Gln Ser
His Gly Lys 50 55 60 Ser Leu Glu Trp Ile Gly Arg Ile Asn Pro Asn
Asn Gly Val Thr Leu 65 70 75 80 Tyr Asn Gln Lys Phe Lys Asp Lys Ala
Ile Leu Thr Val Asp Lys Ser 85 90 95 Ser Thr Thr Ala Tyr Met Glu
Leu Arg Ser Leu Thr Ser Glu Asp Ser 100 105 110 Ala Val Tyr Tyr Cys
Ala Arg Ser Thr Met Ile Thr Asn Tyr Val Met 115 120 125 Asp Tyr Trp
Gly Gln Val Thr Ser Val Thr Val Ser Ser Gly Gly Gly 130 135 140 Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ile Val Met 145 150
155 160 Thr Gln Thr Pro Thr Phe Leu Leu Val Ser Ala Gly Asp Arg Val
Thr 165 170 175 Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val
Ala Trp Tyr 180 185 190 Gln Gln Lys Pro Gly Gln Ser Pro Thr Leu Leu
Ile Ser Tyr Thr Ser 195 200 205 Ser Arg Tyr Ala Gly Val Pro Asp Arg
Phe Ile Gly Ser Gly Tyr Gly 210 215 220 Thr Asp Phe Thr Phe Thr Ile
Ser Thr Leu Gln Ala Glu Asp Leu Ala 225 230 235 240 Val Tyr Phe Cys
Gln Gln Asp Tyr Asn Ser Pro Pro Thr Phe Gly Gly 245 250 255 Gly Thr
Lys Leu Glu Ile Lys Arg Glu Pro Lys Ser Pro Asp Lys Thr 260 265 270
His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 275
280 285 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ala Arg
Thr 290 295 300 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu 305 310 315 320 Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys 325 330 335 Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser 340 345 350 Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 355 360 365 Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 370 375 380 Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 385 390 395
400 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
405 410 415 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn 420 425 430 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser 435 440 445 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg 450 455 460 Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu 465 470 475 480 His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile 485 490 495 Ile Ser Phe
Phe Leu Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu 500 505 510 Phe
Phe Leu Thr Leu Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys 515 520
525 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr
530 535 540 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu
Glu Gly 545 550 555 560 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser
Ala Asp Ala Pro Ala 565 570 575 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
Asn Glu Leu Asn Leu Gly Arg 580 585 590 Arg Glu Glu Tyr Asp Val Leu
Asp Lys Arg Arg Gly Arg Asp Pro Glu 595
600 605 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
Asn 610 615 620 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
Ile Gly Met 625 630 635 640 Lys Gly Glu Arg Arg Arg Gly Lys Gly His
Asp Gly Leu Tyr Gln Gly 645 650 655 Leu Ser Thr Ala Thr Lys Asp Thr
Tyr Asp Ala Leu His Met Gln Ala 660 665 670 Leu Pro Pro Arg 675
25455PRTartificial sequenceA1-v1 polypeptide CAR sequence 25Met Ala
Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15
His Ala Ala Arg Pro Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu 20
25 30 Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr 35 40 45 Thr Phe Thr Asn Phe Gly Met Asn Trp Val Lys Gln Gly
Pro Gly Glu 50 55 60 Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Asn
Thr Gly Glu Pro Arg 65 70 75 80 Tyr Ala Glu Glu Phe Lys Gly Arg Phe
Ala Phe Ser Leu Glu Thr Thr 85 90 95 Ala Ser Thr Ala Tyr Leu Gln
Ile Asn Asn Leu Lys Asn Glu Asp Thr 100 105 110 Ala Thr Tyr Phe Cys
Ala Arg Asp Trp Asp Gly Ala Tyr Phe Phe Asp 115 120 125 Tyr Trp Gly
Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Ser 130 135 140 Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ile Val Met Thr Gln 145 150
155 160 Thr Pro Lys Phe Leu Leu Val Ser Ala Gly Asp Arg Val Thr Ile
Thr 165 170 175 Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala Trp
Tyr Gln Gln 180 185 190 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Asn
Phe Ala Thr Asn Arg 195 200 205 Tyr Thr Gly Val Pro Asn Arg Phe Thr
Gly Ser Gly Tyr Gly Thr Asp 210 215 220 Phe Thr Phe Thr Ile Ser Thr
Val Gln Ala Glu Asp Leu Ala Leu Tyr 225 230 235 240 Phe Cys Gln Gln
Asp Tyr Ser Ser Pro Trp Thr Phe Gly Gly Gly Thr 245 250 255 Lys Leu
Glu Ile Lys Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe 260 265 270
Pro Pro Gly Tyr Gln Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 275
280 285 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg
Gly 290 295 300 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
Arg Pro Val 305 310 315 320 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
Cys Arg Phe Pro Glu Glu 325 330 335 Glu Glu Gly Gly Cys Glu Leu Arg
Val Lys Phe Ser Arg Ser Ala Asp 340 345 350 Ala Pro Ala Tyr Gln Gln
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 355 360 365 Leu Gly Arg Arg
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 370 375 380 Asp Pro
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 385 390 395
400 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
405 410 415 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
Gly Leu 420 425 430 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
Asp Ala Leu His 435 440 445 Met Gln Ala Leu Pro Pro Arg 450 455
26458PRTartificial sequenceA1-v2 polypeptide CAR sequence 26Met Ala
Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15
His Ala Ala Arg Pro Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu 20
25 30 Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr 35 40 45 Thr Phe Thr Asn Phe Gly Met Asn Trp Val Lys Gln Gly
Pro Gly Glu 50 55 60 Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Asn
Thr Gly Glu Pro Arg 65 70 75 80 Tyr Ala Glu Glu Phe Lys Gly Arg Phe
Ala Phe Ser Leu Glu Thr Thr 85 90 95 Ala Ser Thr Ala Tyr Leu Gln
Ile Asn Asn Leu Lys Asn Glu Asp Thr 100 105 110 Ala Thr Tyr Phe Cys
Ala Arg Asp Trp Asp Gly Ala Tyr Phe Phe Asp 115 120 125 Tyr Trp Gly
Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Ser 130 135 140 Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ile Val Met Thr Gln 145 150
155 160 Thr Pro Lys Phe Leu Leu Val Ser Ala Gly Asp Arg Val Thr Ile
Thr 165 170 175 Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala Trp
Tyr Gln Gln 180 185 190 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Asn
Phe Ala Thr Asn Arg 195 200 205 Tyr Thr Gly Val Pro Asn Arg Phe Thr
Gly Ser Gly Tyr Gly Thr Asp 210 215 220 Phe Thr Phe Thr Ile Ser Thr
Val Gln Ala Glu Asp Leu Ala Leu Tyr 225 230 235 240 Phe Cys Gln Gln
Asp Tyr Ser Ser Pro Trp Thr Phe Gly Gly Gly Thr 245 250 255 Lys Leu
Glu Ile Lys Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe 260 265 270
Pro Pro Gly Tyr Gln Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr 275
280 285 Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu Arg Phe Ser Val
Val 290 295 300 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
Pro Phe Met 305 310 315 320 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
Gly Cys Ser Cys Arg Phe 325 330 335 Pro Glu Glu Glu Glu Gly Gly Cys
Glu Leu Arg Val Lys Phe Ser Arg 340 345 350 Ser Ala Asp Ala Pro Ala
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 355 360 365 Glu Leu Asn Leu
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 370 375 380 Arg Gly
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 385 390 395
400 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
405 410 415 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
Gly His 420 425 430 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
Asp Thr Tyr Asp 435 440 445 Ala Leu His Met Gln Ala Leu Pro Pro Arg
450 455 27484PRTartificial sequenceA1-v3 polypeptide CAR sequence
27Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1
5 10 15 His Ala Ala Arg Pro Gln Ile Gln Leu Val Gln Ser Gly Pro Glu
Leu 20 25 30 Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr 35 40 45 Thr Phe Thr Asn Phe Gly Met Asn Trp Val Lys
Gln Gly Pro Gly Glu 50 55 60 Gly Leu Lys Trp Met Gly Trp Ile Asn
Thr Asn Thr Gly Glu Pro Arg 65 70 75 80 Tyr Ala Glu Glu Phe Lys Gly
Arg Phe Ala Phe Ser Leu Glu Thr Thr 85 90 95 Ala Ser Thr Ala Tyr
Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr 100 105 110 Ala Thr Tyr
Phe Cys Ala Arg Asp Trp Asp Gly Ala Tyr Phe Phe Asp 115 120 125 Tyr
Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Ser 130 135
140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ile Val Met Thr Gln
145 150 155 160 Thr Pro Lys Phe Leu Leu Val Ser Ala Gly Asp Arg Val
Thr Ile Thr 165 170 175 Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val
Ala Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Gln Ser Pro Lys Leu Leu
Ile Asn Phe Ala Thr Asn Arg 195 200 205 Tyr Thr Gly Val Pro Asn Arg
Phe Thr Gly Ser Gly Tyr Gly Thr Asp 210 215 220 Phe Thr Phe Thr Ile
Ser Thr Val Gln Ala Glu Asp Leu Ala Leu Tyr 225 230 235 240 Phe Cys
Gln Gln Asp Tyr Ser Ser Pro Trp Thr Phe Gly Gly Gly Thr 245 250 255
Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 260
265 270 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
Cys 275 280 285 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
Asp Phe Ala 290 295 300 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
Thr Cys Gly Val Leu 305 310 315 320 Leu Leu Ser Leu Val Ile Thr Leu
Tyr Cys Lys Arg Gly Arg Lys Lys 325 330 335 Leu Leu Tyr Ile Phe Lys
Gln Pro Phe Met Arg Pro Val Gln Thr Thr 340 345 350 Gln Glu Glu Asp
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 355 360 365 Gly Cys
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 370 375 380
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 385
390 395 400 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
Pro Glu 405 410 415 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
Gly Leu Tyr Asn 420 425 430 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
Tyr Ser Glu Ile Gly Met 435 440 445 Lys Gly Glu Arg Arg Arg Gly Lys
Gly His Asp Gly Leu Tyr Gln Gly 450 455 460 Leu Ser Thr Ala Thr Lys
Asp Thr Tyr Asp Ala Leu His Met Gln Ala 465 470 475 480 Leu Pro Pro
Arg 28487PRTartificial sequenceA1-v4 polypeptide CAR sequence 28Met
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10
15 His Ala Ala Arg Pro Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu
20 25 30 Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr 35 40 45 Thr Phe Thr Asn Phe Gly Met Asn Trp Val Lys Gln
Gly Pro Gly Glu 50 55 60 Gly Leu Lys Trp Met Gly Trp Ile Asn Thr
Asn Thr Gly Glu Pro Arg 65 70 75 80 Tyr Ala Glu Glu Phe Lys Gly Arg
Phe Ala Phe Ser Leu Glu Thr Thr 85 90 95 Ala Ser Thr Ala Tyr Leu
Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr 100 105 110 Ala Thr Tyr Phe
Cys Ala Arg Asp Trp Asp Gly Ala Tyr Phe Phe Asp 115 120 125 Tyr Trp
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Ser 130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ile Val Met Thr Gln 145
150 155 160 Thr Pro Lys Phe Leu Leu Val Ser Ala Gly Asp Arg Val Thr
Ile Thr 165 170 175 Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala
Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
Asn Phe Ala Thr Asn Arg 195 200 205 Tyr Thr Gly Val Pro Asn Arg Phe
Thr Gly Ser Gly Tyr Gly Thr Asp 210 215 220 Phe Thr Phe Thr Ile Ser
Thr Val Gln Ala Glu Asp Leu Ala Leu Tyr 225 230 235 240 Phe Cys Gln
Gln Asp Tyr Ser Ser Pro Trp Thr Phe Gly Gly Gly Thr 245 250 255 Lys
Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 260 265
270 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys
275 280 285 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
Phe Ala 290 295 300 Cys Asp Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser
Thr Ala Leu Leu 305 310 315 320 Phe Leu Leu Phe Phe Leu Thr Leu Arg
Phe Ser Val Val Lys Arg Gly 325 330 335 Arg Lys Lys Leu Leu Tyr Ile
Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350 Gln Thr Thr Gln Glu
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365 Glu Glu Gly
Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390
395 400 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
Arg 405 410 415 Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
Gln Glu Gly 420 425 430 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
Glu Ala Tyr Ser Glu 435 440 445 Ile Gly Met Lys Gly Glu Arg Arg Arg
Gly Lys Gly His Asp Gly Leu 450 455 460 Tyr Gln Gly Leu Ser Thr Ala
Thr Lys Asp Thr Tyr Asp Ala Leu His 465 470 475 480 Met Gln Ala Leu
Pro Pro Arg 485 29670PRTartificial sequenceA1-v5 polypeptide CAR
sequence 29Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu
Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Ile Gln Leu Val Gln Ser
Gly Pro Glu Leu 20 25 30 Lys Lys Pro Gly Glu Thr Val Lys Ile Ser
Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Asn Phe Gly Met Asn
Trp Val Lys Gln Gly Pro Gly Glu 50 55 60 Gly Leu Lys Trp Met Gly
Trp Ile Asn Thr Asn Thr Gly Glu Pro Arg 65 70 75 80 Tyr Ala Glu Glu
Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Thr 85 90 95 Ala Ser
Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr 100 105 110
Ala Thr Tyr Phe Cys Ala Arg Asp Trp Asp Gly Ala Tyr Phe Phe Asp 115
120 125 Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly
Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ile Val
Met Thr Gln 145 150 155 160 Thr Pro Lys Phe Leu Leu Val Ser Ala Gly
Asp Arg Val Thr Ile Thr 165 170 175 Cys Lys Ala Ser Gln Ser Val Ser
Asn Asp Val Ala Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Gln Ser Pro
Lys Leu Leu Ile Asn Phe Ala Thr Asn Arg 195 200 205 Tyr Thr Gly Val
Pro Asn Arg Phe Thr Gly Ser Gly Tyr Gly Thr Asp 210 215 220 Phe Thr
Phe Thr Ile Ser Thr Val Gln Ala Glu Asp Leu Ala Leu Tyr 225 230 235
240 Phe Cys Gln Gln Asp Tyr Ser Ser Pro Trp Thr Phe Gly Gly Gly Thr
245 250 255 Lys Leu Glu Ile Lys Glu Pro Lys Ser Pro Asp Lys Thr His
Thr Cys 260 265 270 Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser
Val Phe Leu Phe 275 280 285 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ala Arg Thr Pro Glu Val 290 295 300 Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe 305
310 315 320 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro 325 330 335 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr 340 345 350 Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val 355 360 365 Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala 370 375 380 Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 385 390 395 400 Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 405 410 415 Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 420 425
430 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
435 440 445 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln 450 455 460 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His 465 470 475 480 Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys Ile Tyr Ile Trp 485 490 495 Ala Pro Leu Ala Gly Thr Cys
Gly Val Leu Leu Leu Ser Leu Val Ile 500 505 510 Thr Leu Tyr Cys Lys
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 515 520 525 Gln Pro Phe
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 530 535 540 Ser
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 545 550
555 560 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
Asn 565 570 575 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
Tyr Asp Val 580 585 590 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
Gly Gly Lys Pro Arg 595 600 605 Arg Lys Asn Pro Gln Glu Gly Leu Tyr
Asn Glu Leu Gln Lys Asp Lys 610 615 620 Met Ala Glu Ala Tyr Ser Glu
Ile Gly Met Lys Gly Glu Arg Arg Arg 625 630 635 640 Gly Lys Gly His
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 645 650 655 Asp Thr
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 660 665 670
30673PRTartificial sequenceA1-v6 polypeptide CAR sequence 30Met Ala
Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15
His Ala Ala Arg Pro Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu 20
25 30 Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr 35 40 45 Thr Phe Thr Asn Phe Gly Met Asn Trp Val Lys Gln Gly
Pro Gly Glu 50 55 60 Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Asn
Thr Gly Glu Pro Arg 65 70 75 80 Tyr Ala Glu Glu Phe Lys Gly Arg Phe
Ala Phe Ser Leu Glu Thr Thr 85 90 95 Ala Ser Thr Ala Tyr Leu Gln
Ile Asn Asn Leu Lys Asn Glu Asp Thr 100 105 110 Ala Thr Tyr Phe Cys
Ala Arg Asp Trp Asp Gly Ala Tyr Phe Phe Asp 115 120 125 Tyr Trp Gly
Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Ser 130 135 140 Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ile Val Met Thr Gln 145 150
155 160 Thr Pro Lys Phe Leu Leu Val Ser Ala Gly Asp Arg Val Thr Ile
Thr 165 170 175 Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val Ala Trp
Tyr Gln Gln 180 185 190 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Asn
Phe Ala Thr Asn Arg 195 200 205 Tyr Thr Gly Val Pro Asn Arg Phe Thr
Gly Ser Gly Tyr Gly Thr Asp 210 215 220 Phe Thr Phe Thr Ile Ser Thr
Val Gln Ala Glu Asp Leu Ala Leu Tyr 225 230 235 240 Phe Cys Gln Gln
Asp Tyr Ser Ser Pro Trp Thr Phe Gly Gly Gly Thr 245 250 255 Lys Leu
Glu Ile Lys Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys 260 265 270
Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe 275
280 285 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ala Arg Thr Pro Glu
Val 290 295 300 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe 305 310 315 320 Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro 325 330 335 Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr 340 345 350 Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 355 360 365 Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 370 375 380 Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 385 390 395
400 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
405 410 415 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro 420 425 430 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser 435 440 445 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln 450 455 460 Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His 465 470 475 480 Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys Ile Ile Ser Phe 485 490 495 Phe Leu Ala
Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu 500 505 510 Thr
Leu Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr 515 520
525 Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
530 535 540 Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
Cys Glu 545 550 555 560 Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
Pro Ala Tyr Gln Gln 565 570 575 Gly Gln Asn Gln Leu Tyr Asn Glu Leu
Asn Leu Gly Arg Arg Glu Glu 580 585 590 Tyr Asp Val Leu Asp Lys Arg
Arg Gly Arg Asp Pro Glu Met Gly Gly 595 600 605 Lys Pro Arg Arg Lys
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 610 615 620 Lys Asp Lys
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 625 630 635 640
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 645
650 655 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
Pro 660 665 670 Arg 31455PRTartificial sequenceA2-v1 polypeptide
CAR sequence 31Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln
Ser Arg Pro Glu Leu 20 25 30 Val Lys Pro Gly Ala Ser Val Lys Met
Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Asp Tyr Val Ile
Ser Trp Val Lys Gln Arg Thr Gly Gln 50 55 60 Gly Leu Glu Trp Ile
Gly Glu Ile Tyr Pro Gly Ser Asn Ser Ile Tyr 65 70 75 80 Tyr Asn Glu
Lys Phe Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser 85 90 95 Ser
Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser 100 105
110 Ala Val Tyr Phe Cys Ala Met Gly Gly Asn Tyr Gly Phe Asp Tyr Trp
115 120 125 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val
Leu Thr Gln Ser 145 150 155 160 Pro Ala Ile Met Ser Ala Ser Leu Gly
Glu Arg Val Thr Leu Thr Cys 165 170 175 Thr Ala Ser Ser Ser Val Asn
Ser Asn Tyr Leu His Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Ser Ser
Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu 195 200 205 Ala Ser Gly
Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser 210 215 220 Tyr
Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr 225 230
235 240 Tyr Cys His Gln Tyr His Arg Ser Pro Leu Thr Phe Gly Ala Gly
Thr 245 250 255 Lys Leu Glu Leu Lys Gly Leu Ala Val Ser Thr Ile Ser
Ser Phe Phe 260 265 270 Pro Pro Gly Tyr Gln Ile Tyr Ile Trp Ala Pro
Leu Ala Gly Thr Cys 275 280 285 Gly Val Leu Leu Leu Ser Leu Val Ile
Thr Leu Tyr Cys Lys Arg Gly 290 295 300 Arg Lys Lys Leu Leu Tyr Ile
Phe Lys Gln Pro Phe Met Arg Pro Val 305 310 315 320 Gln Thr Thr Gln
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 325 330 335 Glu Glu
Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 340 345 350
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 355
360 365 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
Arg 370 375 380 Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
Gln Glu Gly 385 390 395 400 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
Ala Glu Ala Tyr Ser Glu 405 410 415 Ile Gly Met Lys Gly Glu Arg Arg
Arg Gly Lys Gly His Asp Gly Leu 420 425 430 Tyr Gln Gly Leu Ser Thr
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 435 440 445 Met Gln Ala Leu
Pro Pro Arg 450 455 32458PRTartificial sequenceA2-v2 polypeptide
CAR sequence 32Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala
Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln
Ser Arg Pro Glu Leu 20 25 30 Val Lys Pro Gly Ala Ser Val Lys Met
Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Asp Tyr Val Ile
Ser Trp Val Lys Gln Arg Thr Gly Gln 50 55 60 Gly Leu Glu Trp Ile
Gly Glu Ile Tyr Pro Gly Ser Asn Ser Ile Tyr 65 70 75 80 Tyr Asn Glu
Lys Phe Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser 85 90 95 Ser
Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser 100 105
110 Ala Val Tyr Phe Cys Ala Met Gly Gly Asn Tyr Gly Phe Asp Tyr Trp
115 120 125 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val
Leu Thr Gln Ser 145 150 155 160 Pro Ala Ile Met Ser Ala Ser Leu Gly
Glu Arg Val Thr Leu Thr Cys 165 170 175 Thr Ala Ser Ser Ser Val Asn
Ser Asn Tyr Leu His Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Ser Ser
Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu 195 200 205 Ala Ser Gly
Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser 210 215 220 Tyr
Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr 225 230
235 240 Tyr Cys His Gln Tyr His Arg Ser Pro Leu Thr Phe Gly Ala Gly
Thr 245 250 255 Lys Leu Glu Leu Lys Gly Leu Ala Val Ser Thr Ile Ser
Ser Phe Phe 260 265 270 Pro Pro Gly Tyr Gln Ile Ile Ser Phe Phe Leu
Ala Leu Thr Ser Thr 275 280 285 Ala Leu Leu Phe Leu Leu Phe Phe Leu
Thr Leu Arg Phe Ser Val Val 290 295 300 Lys Arg Gly Arg Lys Lys Leu
Leu Tyr Ile Phe Lys Gln Pro Phe Met 305 310 315 320 Arg Pro Val Gln
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 325 330 335 Pro Glu
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 340 345 350
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 355
360 365 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
Arg 370 375 380 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
Lys Asn Pro 385 390 395 400 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
Asp Lys Met Ala Glu Ala 405 410 415 Tyr Ser Glu Ile Gly Met Lys Gly
Glu Arg Arg Arg Gly Lys Gly His 420 425 430 Asp Gly Leu Tyr Gln Gly
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 435 440 445 Ala Leu His Met
Gln Ala Leu Pro Pro Arg 450 455 33484PRTartificial sequenceA2-v3
polypeptide CAR sequence 33Met Ala Leu Pro Val Thr Ala Leu Leu Leu
Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln
Leu Gln Gln Ser Arg Pro Glu Leu 20 25 30 Val Lys Pro Gly Ala Ser
Val Lys Met Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Asp
Tyr Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln 50 55 60 Gly Leu
Glu Trp Ile Gly Glu Ile Tyr Pro Gly Ser Asn Ser Ile Tyr 65 70 75 80
Tyr Asn Glu Lys Phe Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser 85
90 95 Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser 100 105 110 Ala Val Tyr Phe Cys Ala Met Gly Gly Asn Tyr Gly Phe
Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly
Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gln Ile Val Leu Thr Gln Ser 145 150 155 160 Pro Ala Ile Met Ser Ala
Ser Leu Gly Glu Arg Val Thr Leu Thr Cys 165 170 175 Thr Ala Ser Ser
Ser Val Asn Ser Asn Tyr Leu His Trp Tyr Gln Gln 180 185 190 Lys Pro
Gly Ser Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu 195 200 205
Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser 210
215 220 Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr
Tyr 225 230 235 240 Tyr Cys His Gln Tyr His Arg Ser Pro Leu Thr Phe
Gly Ala Gly Thr 245 250 255 Lys Leu Glu Leu Lys Thr Thr Thr Pro Ala
Pro Arg Pro Pro Thr Pro 260 265 270 Ala Pro Thr Ile Ala Ser Gln Pro
Leu Ser Leu Arg Pro Glu Ala Cys 275 280 285 Arg Pro Ala Ala Gly Gly
Ala Val His Thr Arg Gly Leu Asp Phe Ala 290 295 300 Cys Asp Ile Tyr
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 305 310 315 320 Leu
Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 325
330 335 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
Thr 340 345 350 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
Glu Glu Gly 355 360 365 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser
Ala Asp Ala Pro Ala 370 375 380 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
Asn Glu Leu Asn Leu Gly Arg 385 390 395 400 Arg Glu Glu Tyr Asp Val
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 405 410 415 Met Gly Gly Lys
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 420 425 430 Glu Leu
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 435 440 445
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 450
455 460 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
Ala 465 470 475 480 Leu Pro Pro Arg 34487PRTartificial
sequenceA2-v4 polypeptide CAR sequence 34Met Ala Leu Pro Val Thr
Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg
Pro Gln Val Gln Leu Gln Gln Ser Arg Pro Glu Leu 20 25 30 Val Lys
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr 35 40 45
Thr Phe Thr Asp Tyr Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln 50
55 60 Gly Leu Glu Trp Ile Gly Glu Ile Tyr Pro Gly Ser Asn Ser Ile
Tyr 65 70 75 80 Tyr Asn Glu Lys Phe Lys Gly Arg Ala Thr Leu Thr Ala
Asp Lys Ser 85 90 95 Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu
Thr Ser Glu Asp Ser 100 105 110 Ala Val Tyr Phe Cys Ala Met Gly Gly
Asn Tyr Gly Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Thr Leu Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser 145 150 155 160 Pro Ala
Ile Met Ser Ala Ser Leu Gly Glu Arg Val Thr Leu Thr Cys 165 170 175
Thr Ala Ser Ser Ser Val Asn Ser Asn Tyr Leu His Trp Tyr Gln Gln 180
185 190 Lys Pro Gly Ser Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn
Leu 195 200 205 Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser
Gly Thr Ser 210 215 220 Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
Asp Ala Ala Thr Tyr 225 230 235 240 Tyr Cys His Gln Tyr His Arg Ser
Pro Leu Thr Phe Gly Ala Gly Thr 245 250 255 Lys Leu Glu Leu Lys Thr
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 260 265 270 Ala Pro Thr Ile
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 275 280 285 Arg Pro
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 290 295 300
Cys Asp Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu Leu 305
310 315 320 Phe Leu Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val Lys
Arg Gly 325 330 335 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
Met Arg Pro Val 340 345 350 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
Cys Arg Phe Pro Glu Glu 355 360 365 Glu Glu Gly Gly Cys Glu Leu Arg
Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala Pro Ala Tyr Gln Gln
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390 395 400 Leu Gly Arg
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415 Asp
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 420 425
430 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
435 440 445 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
Gly Leu 450 455 460 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
Asp Ala Leu His 465 470 475 480 Met Gln Ala Leu Pro Pro Arg 485
35670PRTartificial sequenceA2-v5 polypeptide CAR sequence 35Met Ala
Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15
His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Ser Arg Pro Glu Leu 20
25 30 Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly
Tyr 35 40 45 Thr Phe Thr Asp Tyr Val Ile Ser Trp Val Lys Gln Arg
Thr Gly Gln 50 55 60 Gly Leu Glu Trp Ile Gly Glu Ile Tyr Pro Gly
Ser Asn Ser Ile Tyr 65 70 75 80 Tyr Asn Glu Lys Phe Lys Gly Arg Ala
Thr Leu Thr Ala Asp Lys Ser 85 90 95 Ser Ser Thr Ala Tyr Met Gln
Leu Ser Ser Leu Thr Ser Glu Asp Ser 100 105 110 Ala Val Tyr Phe Cys
Ala Met Gly Gly Asn Tyr Gly Phe Asp Tyr Trp 115 120 125 Gly Gln Gly
Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser 145 150
155 160 Pro Ala Ile Met Ser Ala Ser Leu Gly Glu Arg Val Thr Leu Thr
Cys 165 170 175 Thr Ala Ser Ser Ser Val Asn Ser Asn Tyr Leu His Trp
Tyr Gln Gln 180 185 190 Lys Pro Gly Ser Ser Pro Lys Leu Trp Ile Tyr
Ser Thr Ser Asn Leu 195 200 205 Ala Ser Gly Val Pro Ala Arg Phe Ser
Gly Ser Gly Ser Gly Thr Ser 210 215 220 Tyr Ser Leu Thr Ile Ser Ser
Met Glu Ala Glu Asp Ala Ala Thr Tyr 225 230 235 240 Tyr Cys His Gln
Tyr His Arg Ser Pro Leu Thr Phe Gly Ala Gly Thr 245 250 255 Lys Leu
Glu Leu Lys Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys 260 265 270
Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe 275
280 285 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ala Arg Thr Pro Glu
Val 290 295 300 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe 305 310 315 320 Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro 325 330 335 Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr 340 345 350 Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 355 360 365 Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 370 375 380 Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 385 390 395
400 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
405 410 415 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro 420 425 430 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser 435 440 445 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln 450 455 460 Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His 465 470 475 480 Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys Ile Tyr Ile Trp 485 490 495 Ala Pro Leu
Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 500 505 510 Thr
Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 515 520
525 Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
530 535 540 Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
Arg Val 545 550 555 560 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
Gln Gln Gly Gln Asn 565 570 575 Gln Leu Tyr Asn Glu Leu Asn Leu Gly
Arg Arg Glu Glu Tyr Asp Val 580 585 590 Leu Asp Lys Arg Arg Gly Arg
Asp Pro Glu Met Gly Gly Lys Pro Arg 595 600 605 Arg Lys Asn Pro Gln
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 610 615 620 Met Ala Glu
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 625 630 635 640
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 645
650 655 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 660
665 670 36673PRTartificial sequenceA2-v6 polypeptide CAR sequence
36Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1
5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Ser Arg Pro Glu
Leu 20 25 30 Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr 35 40 45 Thr Phe Thr Asp Tyr Val Ile Ser Trp Val Lys
Gln Arg Thr Gly Gln 50 55 60 Gly Leu Glu Trp Ile Gly Glu Ile Tyr
Pro Gly Ser Asn Ser Ile Tyr 65 70 75 80 Tyr Asn Glu Lys Phe Lys Gly
Arg Ala Thr Leu Thr Ala Asp Lys Ser 85 90 95 Ser Ser Thr Ala Tyr
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser 100 105 110 Ala Val Tyr
Phe Cys Ala Met Gly Gly Asn Tyr Gly Phe Asp Tyr Trp 115 120 125 Gly
Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 130 135
140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser
145 150 155 160 Pro Ala Ile Met Ser Ala Ser Leu Gly Glu Arg Val Thr
Leu Thr Cys 165 170 175 Thr Ala Ser Ser Ser Val Asn Ser Asn Tyr Leu
His Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Ser Ser Pro Lys Leu Trp
Ile Tyr Ser Thr Ser Asn Leu 195 200 205 Ala Ser Gly Val Pro Ala Arg
Phe Ser Gly Ser Gly Ser Gly Thr Ser 210 215 220 Tyr Ser Leu Thr Ile
Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr 225 230 235 240 Tyr Cys
His Gln Tyr His Arg Ser Pro Leu Thr Phe Gly Ala Gly Thr 245 250 255
Lys Leu Glu Leu Lys Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys 260
265 270 Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu
Phe 275 280 285 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ala Arg Thr
Pro Glu Val 290 295 300 Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe 305 310 315 320 Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro 325 330 335 Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr 340 345 350 Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 355 360 365 Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 370 375 380
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 385
390 395 400 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly 405 410 415 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro 420 425 430 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser 435 440 445 Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln 450 455 460 Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His 465 470 475 480 Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys Ile Ile Ser Phe 485 490 495 Phe
Leu Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu 500 505
510 Thr Leu Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr
515 520 525 Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
Glu Glu 530 535 540 Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
Gly Gly Cys Glu 545 550 555 560 Leu Arg Val Lys Phe Ser Arg Ser Ala
Asp Ala Pro Ala Tyr Gln Gln 565 570 575 Gly Gln Asn Gln Leu Tyr Asn
Glu Leu Asn Leu Gly Arg Arg Glu Glu 580 585 590 Tyr Asp Val Leu Asp
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 595 600 605 Lys Pro Arg
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 610 615 620 Lys
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 625 630
635 640 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
Thr 645 650 655 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
Leu Pro Pro 660 665 670 Arg 37459PRTartificial sequenceA3-v1
polypeptide CAR sequence 37Met Ala Leu Pro Val Thr Ala Leu Leu Leu
Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Pro Lys Gly Ser
Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Asn Thr
Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu
Glu Trp Val Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala 65 70 75 80
Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp 85
90 95 Asp Ser Gln Ser Met Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr
Glu 100 105 110 Asp Thr Ala Met Tyr Tyr Cys Val Arg Gln Trp Asp Tyr
Asp Val Arg 115 120 125 Ala Met Asn Tyr Trp Gly Gln Gly Thr Ser Val
Thr Val Ser Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Asp Ile 145 150 155 160 Val Met Thr Gln Ser His
Ile Phe Met Ser Thr Ser Val Gly Asp Arg 165 170 175 Val Ser Ile Thr
Cys Lys Ala Ser Gln Asp Val Asp Thr Ala Val Ala 180 185 190 Trp Tyr
Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp 195 200 205
Ala Ser Thr Arg Leu Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly 210
215 220 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu
Asp 225 230 235 240 Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr
Pro Tyr Thr Phe 245 250 255 Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
Leu Ala Val Ser Thr Ile 260 265 270 Ser Ser Phe Phe Pro Pro Gly Tyr
Gln Ile Tyr Ile Trp Ala Pro Leu 275 280 285 Ala Gly Thr Cys Gly Val
Leu Leu Leu Ser Leu Val Ile Thr
Leu Tyr 290 295 300 Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
Lys Gln Pro Phe 305 310 315 320 Met Arg Pro Val Gln Thr Thr Gln Glu
Glu Asp Gly Cys Ser Cys Arg 325 330 335 Phe Pro Glu Glu Glu Glu Gly
Gly Cys Glu Leu Arg Val Lys Phe Ser 340 345 350 Arg Ser Ala Asp Ala
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 355 360 365 Asn Glu Leu
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 370 375 380 Arg
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 385 390
395 400 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
Glu 405 410 415 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
Gly Lys Gly 420 425 430 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
Thr Lys Asp Thr Tyr 435 440 445 Asp Ala Leu His Met Gln Ala Leu Pro
Pro Arg 450 455 38462PRTartificial sequenceA3-v2 polypeptide CAR
sequence 38Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu
Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu 20 25 30 Val Gln Pro Lys Gly Ser Leu Lys Leu Ser
Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Asn Thr Tyr Ala Met Asn
Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Ala
Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala 65 70 75 80 Thr Tyr Tyr Ala
Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp 85 90 95 Asp Ser
Gln Ser Met Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu 100 105 110
Asp Thr Ala Met Tyr Tyr Cys Val Arg Gln Trp Asp Tyr Asp Val Arg 115
120 125 Ala Met Asn Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Asp Ile 145 150 155 160 Val Met Thr Gln Ser His Ile Phe Met Ser
Thr Ser Val Gly Asp Arg 165 170 175 Val Ser Ile Thr Cys Lys Ala Ser
Gln Asp Val Asp Thr Ala Val Ala 180 185 190 Trp Tyr Gln Gln Lys Pro
Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp 195 200 205 Ala Ser Thr Arg
Leu Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly 210 215 220 Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp 225 230 235
240 Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr Thr Phe
245 250 255 Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Leu Ala Val Ser
Thr Ile 260 265 270 Ser Ser Phe Phe Pro Pro Gly Tyr Gln Ile Ile Ser
Phe Phe Leu Ala 275 280 285 Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu
Phe Phe Leu Thr Leu Arg 290 295 300 Phe Ser Val Val Lys Arg Gly Arg
Lys Lys Leu Leu Tyr Ile Phe Lys 305 310 315 320 Gln Pro Phe Met Arg
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 325 330 335 Ser Cys Arg
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 340 345 350 Lys
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 355 360
365 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
370 375 380 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
Pro Arg 385 390 395 400 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
Leu Gln Lys Asp Lys 405 410 415 Met Ala Glu Ala Tyr Ser Glu Ile Gly
Met Lys Gly Glu Arg Arg Arg 420 425 430 Gly Lys Gly His Asp Gly Leu
Tyr Gln Gly Leu Ser Thr Ala Thr Lys 435 440 445 Asp Thr Tyr Asp Ala
Leu His Met Gln Ala Leu Pro Pro Arg 450 455 460 39488PRTartificial
sequenceA3-v3 polypeptide CAR sequence 39Met Ala Leu Pro Val Thr
Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg
Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln
Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45
Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys 50
55 60 Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr
Ala 65 70 75 80 Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile
Ser Arg Asp 85 90 95 Asp Ser Gln Ser Met Leu Tyr Leu Gln Met Asn
Asn Leu Lys Thr Glu 100 105 110 Asp Thr Ala Met Tyr Tyr Cys Val Arg
Gln Trp Asp Tyr Asp Val Arg 115 120 125 Ala Met Asn Tyr Trp Gly Gln
Gly Thr Ser Val Thr Val Ser Ser Gly 130 135 140 Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 145 150 155 160 Val Met
Thr Gln Ser His Ile Phe Met Ser Thr Ser Val Gly Asp Arg 165 170 175
Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Asp Thr Ala Val Ala 180
185 190 Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr
Trp 195 200 205 Ala Ser Thr Arg Leu Thr Gly Val Pro Asp Arg Phe Thr
Gly Ser Gly 210 215 220 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn
Val Gln Ser Glu Asp 225 230 235 240 Leu Ala Asp Tyr Phe Cys Gln Gln
Tyr Ser Ser Tyr Pro Tyr Thr Phe 245 250 255 Gly Gly Gly Thr Lys Leu
Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg 260 265 270 Pro Pro Thr Pro
Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 275 280 285 Pro Glu
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 290 295 300
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 305
310 315 320 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
Lys Arg 325 330 335 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
Phe Met Arg Pro 340 345 350 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
Ser Cys Arg Phe Pro Glu 355 360 365 Glu Glu Glu Gly Gly Cys Glu Leu
Arg Val Lys Phe Ser Arg Ser Ala 370 375 380 Asp Ala Pro Ala Tyr Gln
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 385 390 395 400 Asn Leu Gly
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 405 410 415 Arg
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 420 425
430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg 485
40491PRTartificial sequenceA3-v4 polypeptide CAR sequence 40Met Ala
Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 20
25 30 Val Gln Pro Lys Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly
Phe 35 40 45 Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala
Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys
Ser Asn Asn Tyr Ala 65 70 75 80 Thr Tyr Tyr Ala Asp Ser Val Lys Asp
Arg Phe Thr Ile Ser Arg Asp 85 90 95 Asp Ser Gln Ser Met Leu Tyr
Leu Gln Met Asn Asn Leu Lys Thr Glu 100 105 110 Asp Thr Ala Met Tyr
Tyr Cys Val Arg Gln Trp Asp Tyr Asp Val Arg 115 120 125 Ala Met Asn
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly 130 135 140 Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 145 150
155 160 Val Met Thr Gln Ser His Ile Phe Met Ser Thr Ser Val Gly Asp
Arg 165 170 175 Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Asp Thr
Ala Val Ala 180 185 190 Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys
Leu Leu Ile Tyr Trp 195 200 205 Ala Ser Thr Arg Leu Thr Gly Val Pro
Asp Arg Phe Thr Gly Ser Gly 210 215 220 Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Asn Val Gln Ser Glu Asp 225 230 235 240 Leu Ala Asp Tyr
Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr Thr Phe 245 250 255 Gly Gly
Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg 260 265 270
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 275
280 285 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg
Gly 290 295 300 Leu Asp Phe Ala Cys Asp Ile Ile Ser Phe Phe Leu Ala
Leu Thr Ser 305 310 315 320 Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu
Thr Leu Arg Phe Ser Val 325 330 335 Val Lys Arg Gly Arg Lys Lys Leu
Leu Tyr Ile Phe Lys Gln Pro Phe 340 345 350 Met Arg Pro Val Gln Thr
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 355 360 365 Phe Pro Glu Glu
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 370 375 380 Arg Ser
Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 385 390 395
400 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
405 410 415 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
Lys Asn 420 425 430 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
Lys Met Ala Glu 435 440 445 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
Arg Arg Arg Gly Lys Gly 450 455 460 His Asp Gly Leu Tyr Gln Gly Leu
Ser Thr Ala Thr Lys Asp Thr Tyr 465 470 475 480 Asp Ala Leu His Met
Gln Ala Leu Pro Pro Arg 485 490 41674PRTartificial sequenceA3-v5
polypeptide CAR sequence 41Met Ala Leu Pro Val Thr Ala Leu Leu Leu
Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Pro Lys Gly Ser
Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Asn Thr
Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu
Glu Trp Val Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala 65 70 75 80
Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp 85
90 95 Asp Ser Gln Ser Met Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr
Glu 100 105 110 Asp Thr Ala Met Tyr Tyr Cys Val Arg Gln Trp Asp Tyr
Asp Val Arg 115 120 125 Ala Met Asn Tyr Trp Gly Gln Gly Thr Ser Val
Thr Val Ser Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Asp Ile 145 150 155 160 Val Met Thr Gln Ser His
Ile Phe Met Ser Thr Ser Val Gly Asp Arg 165 170 175 Val Ser Ile Thr
Cys Lys Ala Ser Gln Asp Val Asp Thr Ala Val Ala 180 185 190 Trp Tyr
Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp 195 200 205
Ala Ser Thr Arg Leu Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly 210
215 220 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu
Asp 225 230 235 240 Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr
Pro Tyr Thr Phe 245 250 255 Gly Gly Gly Thr Lys Leu Glu Ile Lys Glu
Pro Lys Ser Pro Asp Lys 260 265 270 Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Pro Val Ala Gly Pro Ser 275 280 285 Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ala Arg 290 295 300 Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 305 310 315 320 Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 325 330
335 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
340 345 350 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr 355 360 365 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 370 375 380 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu 385 390 395 400 Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys 405 410 415 Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 420 425 430 Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 435 440 445 Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 450 455
460 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
465 470 475 480 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 485 490 495 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
Gly Val Leu Leu Leu 500 505 510 Ser Leu Val Ile Thr Leu Tyr Cys Lys
Arg Gly Arg Lys Lys Leu Leu 515 520 525 Tyr Ile Phe Lys Gln Pro Phe
Met Arg Pro Val Gln Thr Thr Gln Glu 530 535 540 Glu Asp Gly Cys Ser
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 545 550 555 560 Glu Leu
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 565 570 575
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 580
585 590 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
Gly 595 600 605 Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
Asn Glu Leu 610 615 620 Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
Ile Gly Met Lys Gly 625 630 635 640 Glu Arg Arg Arg Gly Lys Gly His
Asp Gly Leu Tyr Gln Gly Leu Ser 645 650 655 Thr Ala Thr Lys Asp Thr
Tyr Asp Ala Leu His Met Gln Ala Leu Pro 660 665
670 Pro Arg 42677PRTartificial sequenceA3-v6 polypeptide CAR
sequence 42Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu
Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu 20 25 30 Val Gln Pro Lys Gly Ser Leu Lys Leu Ser
Cys Ala Ala Ser Gly Phe 35 40 45 Thr Phe Asn Thr Tyr Ala Met Asn
Trp Val Arg Gln Ala Pro Gly Lys 50 55 60 Gly Leu Glu Trp Val Ala
Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala 65 70 75 80 Thr Tyr Tyr Ala
Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp 85 90 95 Asp Ser
Gln Ser Met Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu 100 105 110
Asp Thr Ala Met Tyr Tyr Cys Val Arg Gln Trp Asp Tyr Asp Val Arg 115
120 125 Ala Met Asn Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Asp Ile 145 150 155 160 Val Met Thr Gln Ser His Ile Phe Met Ser
Thr Ser Val Gly Asp Arg 165 170 175 Val Ser Ile Thr Cys Lys Ala Ser
Gln Asp Val Asp Thr Ala Val Ala 180 185 190 Trp Tyr Gln Gln Lys Pro
Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp 195 200 205 Ala Ser Thr Arg
Leu Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly 210 215 220 Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser Glu Asp 225 230 235
240 Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Tyr Thr Phe
245 250 255 Gly Gly Gly Thr Lys Leu Glu Ile Lys Glu Pro Lys Ser Pro
Asp Lys 260 265 270 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val
Ala Gly Pro Ser 275 280 285 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ala Arg 290 295 300 Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro 305 310 315 320 Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 325 330 335 Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 340 345 350 Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 355 360
365 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
370 375 380 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 385 390 395 400 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys 405 410 415 Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 420 425 430 Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp 435 440 445 Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 450 455 460 Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 465 470 475 480
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 485
490 495 Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu Leu Phe
Leu 500 505 510 Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val Lys Arg
Gly Arg Lys 515 520 525 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
Arg Pro Val Gln Thr 530 535 540 Thr Gln Glu Glu Asp Gly Cys Ser Cys
Arg Phe Pro Glu Glu Glu Glu 545 550 555 560 Gly Gly Cys Glu Leu Arg
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 565 570 575 Ala Tyr Gln Gln
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 580 585 590 Arg Arg
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 595 600 605
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 610
615 620 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
Gly 625 630 635 640 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
Gly Leu Tyr Gln 645 650 655 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
Asp Ala Leu His Met Gln 660 665 670 Ala Leu Pro Pro Arg 675
43530PRTartificial sequenceTAL binding domain TRAC_T01-L 43Leu Thr
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys 1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20
25 30 His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn
Gly 35 40 45 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
Val Leu Cys 50 55 60 Gln Ala His Gly Leu Thr Pro Gln Gln Val Val
Ala Ile Ala Ser Asn 65 70 75 80 Gly Gly Gly Lys Gln Ala Leu Glu Thr
Val Gln Arg Leu Leu Pro Val 85 90 95 Leu Cys Gln Ala His Gly Leu
Thr Pro Glu Gln Val Val Ala Ile Ala 100 105 110 Ser His Asp Gly Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 115 120 125 Pro Val Leu
Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 130 135 140 Ile
Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 145 150
155 160 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln
Val 165 170 175 Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
Glu Thr Val 180 185 190 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly Leu Thr Pro Glu 195 200 205 Gln Val Val Ala Ile Ala Ser Asn Ile
Gly Gly Lys Gln Ala Leu Glu 210 215 220 Thr Val Gln Ala Leu Leu Pro
Val Leu Cys Gln Ala His Gly Leu Thr 225 230 235 240 Pro Glu Gln Val
Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala 245 250 255 Leu Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 260 265 270
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys 275
280 285 Gln Ala Leu Glu Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln
Ala 290 295 300 His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser
Asn Asn Gly 305 310 315 320 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys 325 330 335 Gln Ala His Gly Leu Thr Pro Glu
Gln Val Val Ala Ile Ala Ser Asn 340 345 350 Ile Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Ala Leu Leu Pro Val 355 360 365 Leu Cys Gln Ala
His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala 370 375 380 Ser Asn
Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 385 390 395
400 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Ala 420 425 430 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
Pro Gln Gln Val 435 440 445 Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
Gln Ala Leu Glu Thr Val 450 455 460 Gln Arg Leu Leu Pro Val Leu Cys
Gln Ala His Gly Leu Thr Pro Glu 465 470 475 480 Gln Val Val Ala Ile
Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu 485 490 495 Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 500 505 510 Pro
Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala 515 520
525 Leu Glu 530 442814DNAartificial sequencepolynucleotide encoding
TRAC_T01-L TALEN 44atgggcgatc ctaaaaagaa acgtaaggtc atcgattacc
catacgatgt tccagattac 60gctatcgata tcgccgatct acgcacgctc ggctacagcc
agcagcaaca ggagaagatc 120aaaccgaagg ttcgttcgac agtggcgcag
caccacgagg cactggtcgg ccacgggttt 180acacacgcgc acatcgttgc
gttaagccaa cacccggcag cgttagggac cgtcgctgtc 240aagtatcagg
acatgatcgc agcgttgcca gaggcgacac acgaagcgat cgttggcgtc
300ggcaaacagt ggtccggcgc acgcgctctg gaggccttgc tcacggtggc
gggagagttg 360agaggtccac cgttacagtt ggacacaggc caacttctca
agattgcaaa acgtggcggc 420gtgaccgcag tggaggcagt gcatgcatgg
cgcaatgcac tgacgggtgc cccgctcaac 480ttgacccccc agcaggtggt
ggccatcgcc agcaatggcg gtggcaagca ggcgctggag 540acggtccagc
ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ccagcaggtg
600gtggccatcg ccagcaataa tggtggcaag caggcgctgg agacggtcca
gcggctgttg 660ccggtgctgt gccaggccca cggcttgacc ccccagcagg
tggtggccat cgccagcaat 720ggcggtggca agcaggcgct ggagacggtc
cagcggctgt tgccggtgct gtgccaggcc 780cacggcttga ccccggagca
ggtggtggcc atcgccagcc acgatggcgg caagcaggcg 840ctggagacgg
tccagcggct gttgccggtg ctgtgccagg cccacggctt gaccccggag
900caggtggtgg ccatcgccag ccacgatggc ggcaagcagg cgctggagac
ggtccagcgg 960ctgttgccgg tgctgtgcca ggcccacggc ttgaccccgg
agcaggtggt ggccatcgcc 1020agccacgatg gcggcaagca ggcgctggag
acggtccagc ggctgttgcc ggtgctgtgc 1080caggcccacg gcttgacccc
ggagcaggtg gtggccatcg ccagcaatat tggtggcaag 1140caggcgctgg
agacggtgca ggcgctgttg ccggtgctgt gccaggccca cggcttgacc
1200ccggagcagg tggtggccat cgccagccac gatggcggca agcaggcgct
ggagacggtc 1260cagcggctgt tgccggtgct gtgccaggcc cacggcttga
ccccggagca ggtggtggcc 1320atcgccagca atattggtgg caagcaggcg
ctggagacgg tgcaggcgct gttgccggtg 1380ctgtgccagg cccacggctt
gaccccccag caggtggtgg ccatcgccag caataatggt 1440ggcaagcagg
cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca ggcccacggc
1500ttgaccccgg agcaggtggt ggccatcgcc agcaatattg gtggcaagca
ggcgctggag 1560acggtgcagg cgctgttgcc ggtgctgtgc caggcccacg
gcttgacccc ccagcaggtg 1620gtggccatcg ccagcaatgg cggtggcaag
caggcgctgg agacggtcca gcggctgttg 1680ccggtgctgt gccaggccca
cggcttgacc ccggagcagg tggtggccat cgccagcaat 1740attggtggca
agcaggcgct ggagacggtg caggcgctgt tgccggtgct gtgccaggcc
1800cacggcttga ccccccagca ggtggtggcc atcgccagca atggcggtgg
caagcaggcg 1860ctggagacgg tccagcggct gttgccggtg ctgtgccagg
cccacggctt gaccccggag 1920caggtggtgg ccatcgccag ccacgatggc
ggcaagcagg cgctggagac ggtccagcgg 1980ctgttgccgg tgctgtgcca
ggcccacggc ttgacccctc agcaggtggt ggccatcgcc 2040agcaatggcg
gcggcaggcc ggcgctggag agcattgttg cccagttatc tcgccctgat
2100ccggcgttgg ccgcgttgac caacgaccac ctcgtcgcct tggcctgcct
cggcgggcgt 2160cctgcgctgg atgcagtgaa aaagggattg ggggatccta
tcagccgttc ccagctggtg 2220aagtccgagc tggaggagaa gaaatccgag
ttgaggcaca agctgaagta cgtgccccac 2280gagtacatcg agctgatcga
gatcgcccgg aacagcaccc aggaccgtat cctggagatg 2340aaggtgatgg
agttcttcat gaaggtgtac ggctacaggg gcaagcacct gggcggctcc
2400aggaagcccg acggcgccat ctacaccgtg ggctccccca tcgactacgg
cgtgatcgtg 2460gacaccaagg cctactccgg cggctacaac ctgcccatcg
gccaggccga cgaaatgcag 2520aggtacgtgg aggagaacca gaccaggaac
aagcacatca accccaacga gtggtggaag 2580gtgtacccct ccagcgtgac
cgagttcaag ttcctgttcg tgtccggcca cttcaagggc 2640aactacaagg
cccagctgac caggctgaac cacatcacca actgcaacgg cgccgtgctg
2700tccgtggagg agctcctgat cggcggcgag atgatcaagg ccggcaccct
gaccctggag 2760gaggtgagga ggaagttcaa caacggcgag atcaacttcg
cggccgactg ataa 2814452832DNAartificial sequencepolynucleotide
encoding TRAC_T01-R TALEN 45atgggcgatc ctaaaaagaa acgtaaggtc
atcgataagg agaccgccgc tgccaagttc 60gagagacagc acatggacag catcgatatc
gccgatctac gcacgctcgg ctacagccag 120cagcaacagg agaagatcaa
accgaaggtt cgttcgacag tggcgcagca ccacgaggca 180ctggtcggcc
acgggtttac acacgcgcac atcgttgcgt taagccaaca cccggcagcg
240ttagggaccg tcgctgtcaa gtatcaggac atgatcgcag cgttgccaga
ggcgacacac 300gaagcgatcg ttggcgtcgg caaacagtgg tccggcgcac
gcgctctgga ggccttgctc 360acggtggcgg gagagttgag aggtccaccg
ttacagttgg acacaggcca acttctcaag 420attgcaaaac gtggcggcgt
gaccgcagtg gaggcagtgc atgcatggcg caatgcactg 480acgggtgccc
cgctcaactt gaccccggag caggtggtgg ccatcgccag ccacgatggc
540ggcaagcagg cgctggagac ggtccagcgg ctgttgccgg tgctgtgcca
ggcccacggc 600ttgacccccc agcaggtggt ggccatcgcc agcaatggcg
gtggcaagca ggcgctggag 660acggtccagc ggctgttgcc ggtgctgtgc
caggcccacg gcttgacccc ggagcaggtg 720gtggccatcg ccagccacga
tggcggcaag caggcgctgg agacggtcca gcggctgttg 780ccggtgctgt
gccaggccca cggcttgacc ccggagcagg tggtggccat cgccagcaat
840attggtggca agcaggcgct ggagacggtg caggcgctgt tgccggtgct
gtgccaggcc 900cacggcttga ccccccagca ggtggtggcc atcgccagca
ataatggtgg caagcaggcg 960ctggagacgg tccagcggct gttgccggtg
ctgtgccagg cccacggctt gaccccggag 1020caggtggtgg ccatcgccag
ccacgatggc ggcaagcagg cgctggagac ggtccagcgg 1080ctgttgccgg
tgctgtgcca ggcccacggc ttgacccccc agcaggtggt ggccatcgcc
1140agcaatggcg gtggcaagca ggcgctggag acggtccagc ggctgttgcc
ggtgctgtgc 1200caggcccacg gcttgacccc ccagcaggtg gtggccatcg
ccagcaataa tggtggcaag 1260caggcgctgg agacggtcca gcggctgttg
ccggtgctgt gccaggccca cggcttgacc 1320ccccagcagg tggtggccat
cgccagcaat aatggtggca agcaggcgct ggagacggtc 1380cagcggctgt
tgccggtgct gtgccaggcc cacggcttga ccccccagca ggtggtggcc
1440atcgccagca atggcggtgg caagcaggcg ctggagacgg tccagcggct
gttgccggtg 1500ctgtgccagg cccacggctt gaccccggag caggtggtgg
ccatcgccag caatattggt 1560ggcaagcagg cgctggagac ggtgcaggcg
ctgttgccgg tgctgtgcca ggcccacggc 1620ttgaccccgg agcaggtggt
ggccatcgcc agccacgatg gcggcaagca ggcgctggag 1680acggtccagc
ggctgttgcc ggtgctgtgc caggcccacg gcttgacccc ggagcaggtg
1740gtggccatcg ccagcaatat tggtggcaag caggcgctgg agacggtgca
ggcgctgttg 1800ccggtgctgt gccaggccca cggcttgacc ccggagcagg
tggtggccat cgccagccac 1860gatggcggca agcaggcgct ggagacggtc
cagcggctgt tgccggtgct gtgccaggcc 1920cacggcttga ccccccagca
ggtggtggcc atcgccagca ataatggtgg caagcaggcg 1980ctggagacgg
tccagcggct gttgccggtg ctgtgccagg cccacggctt gacccctcag
2040caggtggtgg ccatcgccag caatggcggc ggcaggccgg cgctggagag
cattgttgcc 2100cagttatctc gccctgatcc ggcgttggcc gcgttgacca
acgaccacct cgtcgccttg 2160gcctgcctcg gcgggcgtcc tgcgctggat
gcagtgaaaa agggattggg ggatcctatc 2220agccgttccc agctggtgaa
gtccgagctg gaggagaaga aatccgagtt gaggcacaag 2280ctgaagtacg
tgccccacga gtacatcgag ctgatcgaga tcgcccggaa cagcacccag
2340gaccgtatcc tggagatgaa ggtgatggag ttcttcatga aggtgtacgg
ctacaggggc 2400aagcacctgg gcggctccag gaagcccgac ggcgccatct
acaccgtggg ctcccccatc 2460gactacggcg tgatcgtgga caccaaggcc
tactccggcg gctacaacct gcccatcggc 2520caggccgacg aaatgcagag
gtacgtggag gagaaccaga ccaggaacaa gcacatcaac 2580cccaacgagt
ggtggaaggt gtacccctcc agcgtgaccg agttcaagtt cctgttcgtg
2640tccggccact tcaagggcaa ctacaaggcc cagctgacca ggctgaacca
catcaccaac 2700tgcaacggcg ccgtgctgtc cgtggaggag ctcctgatcg
gcggcgagat gatcaaggcc 2760ggcaccctga ccctggagga ggtgaggagg
aagttcaaca acggcgagat caacttcgcg 2820gccgactgat aa
283246530PRTartificial sequenceTAL binding domain TRAC_T01-L 46Leu
Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys 1 5 10
15 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30 His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn
Asn Gly 35 40 45 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
Pro Val Leu Cys 50 55 60 Gln Ala His Gly Leu Thr Pro Gln Gln Val
Val Ala Ile Ala Ser Asn 65 70 75 80 Gly Gly Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu Pro Val 85 90 95 Leu Cys Gln Ala His Gly
Leu Thr Pro Glu Gln Val Val Ala Ile Ala 100 105 110 Ser His Asp Gly
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 115 120 125 Pro Val
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala 130 135 140
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 145
150 155 160 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
Gln Val 165 170 175 Val Ala Ile Ala
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val 180 185 190 Gln Arg
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu 195 200 205
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu 210
215 220 Thr Val Gln Ala Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu
Thr 225 230 235 240 Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
Gly Lys Gln Ala 245 250 255 Leu Glu Thr Val Gln Arg Leu Leu Pro Val
Leu Cys Gln Ala His Gly 260 265 270 Leu Thr Pro Glu Gln Val Val Ala
Ile Ala Ser Asn Ile Gly Gly Lys 275 280 285 Gln Ala Leu Glu Thr Val
Gln Ala Leu Leu Pro Val Leu Cys Gln Ala 290 295 300 His Gly Leu Thr
Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly 305 310 315 320 Gly
Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys 325 330
335 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
340 345 350 Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu
Pro Val 355 360 365 Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val
Val Ala Ile Ala 370 375 380 Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu
Thr Val Gln Arg Leu Leu 385 390 395 400 Pro Val Leu Cys Gln Ala His
Gly Leu Thr Pro Glu Gln Val Val Ala 405 410 415 Ile Ala Ser Asn Ile
Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Ala 420 425 430 Leu Leu Pro
Val Leu Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val 435 440 445 Val
Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val 450 455
460 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480 Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln
Ala Leu Glu 485 490 495 Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Ala His Gly Leu Thr 500 505 510 Pro Gln Gln Val Val Ala Ile Ala Ser
Asn Gly Gly Gly Arg Pro Ala 515 520 525 Leu Glu 530
47530PRTartificial sequenceTAL binding domain TRAC_T01-R 47Leu Thr
Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys 1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20
25 30 His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Gly
Gly 35 40 45 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro
Val Leu Cys 50 55 60 Gln Ala His Gly Leu Thr Pro Glu Gln Val Val
Ala Ile Ala Ser His 65 70 75 80 Asp Gly Gly Lys Gln Ala Leu Glu Thr
Val Gln Arg Leu Leu Pro Val 85 90 95 Leu Cys Gln Ala His Gly Leu
Thr Pro Glu Gln Val Val Ala Ile Ala 100 105 110 Ser Asn Ile Gly Gly
Lys Gln Ala Leu Glu Thr Val Gln Ala Leu Leu 115 120 125 Pro Val Leu
Cys Gln Ala His Gly Leu Thr Pro Gln Gln Val Val Ala 130 135 140 Ile
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg 145 150
155 160 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln
Val 165 170 175 Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu
Glu Thr Val 180 185 190 Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His
Gly Leu Thr Pro Gln 195 200 205 Gln Val Val Ala Ile Ala Ser Asn Gly
Gly Gly Lys Gln Ala Leu Glu 210 215 220 Thr Val Gln Arg Leu Leu Pro
Val Leu Cys Gln Ala His Gly Leu Thr 225 230 235 240 Pro Gln Gln Val
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala 245 250 255 Leu Glu
Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly 260 265 270
Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys 275
280 285 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln
Ala 290 295 300 His Gly Leu Thr Pro Gln Gln Val Val Ala Ile Ala Ser
Asn Gly Gly 305 310 315 320 Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
Leu Leu Pro Val Leu Cys 325 330 335 Gln Ala His Gly Leu Thr Pro Glu
Gln Val Val Ala Ile Ala Ser Asn 340 345 350 Ile Gly Gly Lys Gln Ala
Leu Glu Thr Val Gln Ala Leu Leu Pro Val 355 360 365 Leu Cys Gln Ala
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala 370 375 380 Ser His
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu 385 390 395
400 Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415 Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val
Gln Ala 420 425 430 Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
Pro Glu Gln Val 435 440 445 Val Ala Ile Ala Ser His Asp Gly Gly Lys
Gln Ala Leu Glu Thr Val 450 455 460 Gln Arg Leu Leu Pro Val Leu Cys
Gln Ala His Gly Leu Thr Pro Gln 465 470 475 480 Gln Val Val Ala Ile
Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu 485 490 495 Thr Val Gln
Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr 500 505 510 Pro
Gln Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg Pro Ala 515 520
525 Leu Glu 530 488PRTartificial sequenceCDR1 of H8 VH chain 48Gly
Tyr Ser Phe Thr Gly Tyr Tyr 1 5 498PRTartificial sequenceCDR2 of H8
VH chain 49Ile Asn Pro Asn Asn Gly Val Thr 1 5 5013PRTartificial
sequenceCDR3 of H8 VH chain 50Ala Arg Ser Thr Met Ile Thr Asn Tyr
Val Met Asp Tyr 1 5 10 516PRTartificial sequenceCDR1 of H8 VL chain
51Gln Ser Val Ser Asn Asp 1 5 523PRTartificial sequenceCDR2 of H8
VL chain 52Tyr Thr Ser 1 539PRTartificial sequenceCDR3 of H8 VL
chain 53Gln Gln Asp Tyr Asn Ser Pro Pro Thr 1 5 548PRTartificial
sequenceCDR1 of A1 VH chain 54Gly Tyr Thr Phe Thr Asn Phe Gly 1 5
558PRTartificial sequenceCDR2 of A1 VH chain 55Ile Asn Thr Asn Thr
Gly Glu Pro 1 5 5612PRTartificial sequenceCDR3 of A1 VH chain 56Ala
Arg Asp Trp Asp Gly Ala Tyr Phe Phe Asp Tyr 1 5 10 576PRTartificial
sequenceCDR1 of A1 VL chain 57Gln Ser Val Ser Asn Asp 1 5
583PRTartificial sequenceCDR2 of A1 VL chain 58Phe Ala Thr 1
599PRTartificial sequenceCDR3 of A1 VL chain 59Gln Gln Asp Tyr Ser
Ser Pro Trp Thr 1 5 608PRTartificial sequenceCDR1 of A2 VH chain
60Gly Tyr Thr Phe Thr Asp Tyr Val 1 5 618PRTartificial sequenceCDR2
of A2 VH chain 61Ile Tyr Pro Gly Ser Asn Ser Ile 1 5
6210PRTartificial sequenceCDR3 of A2 VH chain 62Ala Met Gly Gly Asn
Tyr Gly Phe Asp Tyr 1 5 10 637PRTartificial sequenceCDR1 of A2 VL
chain 63Ser Ser Val Asn Ser Asn Tyr 1 5 643PRTartificial
sequenceCDR2 of A2 VL chain 64Ser Thr Ser 1 659PRTartificial
sequenceCDR3 of A2 VL chain 65His Gln Tyr His Arg Ser Pro Leu Thr 1
5 668PRTartificial sequenceCDR1 of A3 VH chain 66Gly Phe Thr Phe
Asn Thr Tyr Ala 1 5 6710PRTartificial sequenceCDR2 of A3 VH chain
67Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr 1 5 10 6813PRTartificial
sequenceCDR3 of A3 VH chain 68Val Arg Gln Trp Asp Tyr Asp Val Arg
Ala Met Asn Tyr 1 5 10 696PRTartificial sequenceCDR1 of A3 VL chain
69Gln Asp Val Asp Thr Ala 1 5 703PRTartificial sequenceCDR2 of A3
VL chain 70Trp Ala Ser 1 719PRTartificial sequenceCDR3 of A3 VL
chain 71Gln Gln Tyr Ser Ser Tyr Pro Tyr Thr 1 5
* * * * *
References